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NanoLuc Complementation Reporter Optimized for Accurate


Measurement of Protein Interactions in Cells
Andrew S. Dixon,†,§ Marie K. Schwinn,† Mary P. Hall,† Kris Zimmerman,† Paul Otto,†
Thomas H. Lubben,† Braeden L. Butler,† Brock F. Binkowski,† Thomas Machleidt,† Thomas A. Kirkland,‡
Monika G. Wood,† Christopher T. Eggers,† Lance P. Encell,*,† and Keith V. Wood†

Promega Corporation, Madison, Wisconsin 53711, United States

Promega Biosciences Incorporated, San Luis Obispo, California 93401, United States
*
S Supporting Information
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ABSTRACT: Protein-fragment complementation assays (PCAs) are


widely used for investigating protein interactions. However, the
Downloaded via 83.132.123.215 on September 20, 2019 at 14:49:37 (UTC).

fragments used are structurally compromised and have not been


optimized nor thoroughly characterized for accurately assessing these
interactions. We took advantage of the small size and bright
luminescence of NanoLuc to engineer a new complementation reporter
(NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18
kDa polypeptide) weakly associate so that their assembly into a
luminescent complex is dictated by the interaction characteristics of the
target proteins onto which they are appended. To ascertain their
general suitability for measuring interaction affinities and kinetics, we
determined that their intrinsic affinity (KD = 190 μM) and association
constants (kon = 500 M−1 s−1, koff = 0.2 s−1) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT
was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and
a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear
characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible,
and robust to temperature (21−37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors
known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT
allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.

P roteins are central to the physiological dynamics of living


cells, forming interactive networks that are responsive to
evolving circumstances (e.g., environmental conditions, stress or
(116 kDa monomer) has also been used as a split reporter,4 but
its size and requirement for oligomerization further impose
interaction relationships that may be inconsistent with the target
pathological situations, or the action of drugs).1 The interactions proteins. Furthermore, reporters such as β-galactosidase that rely
arise through a complex interplay of structural conformations, on the accumulation of an enzymatic product for detection are
relationships with other molecules, and microenvironmental hindered in their ability to reveal process dynamics.
surroundings. Due to the complexity of these functionally Assays based on split luciferases are often preferred due to
contingent ensembles, analytical methods are needed that do not their sensitivity and simplicity. Luminescence provides an
disturb the native cellular context. Split reporters have been instantaneous indication of reporter activity in living cells and
commonly used for this purpose, where complementary reporter is readily generated by adding substrate to the growth medium.
fragments are genetically fused onto a pair of interacting However, although commonly regarded for producing quanti-
proteins.2 The fragments normally exhibit little reporter activity tative data, split luciferases have been evaluated largely for assay
separately, but regain activity when brought together through the
precision rather than accuracy.5−8 While reproducibility and
interaction of their fused partners.
correlation with known behaviors of interacting proteins have
Although conceptually straightforward, the ability of split
reporters to provide quantitative data without perturbing the been shown, quantitative concordance of assay data with the
underlying interaction dynamics is not assured. Because underlying molecular interaction has been difficult to substan-
association of the reporter fragments is required for the tiate. Hence, reinforced by the known limitations associated with
reconstituted activity, the intrinsic binding affinity of the many split reporters, doubts persist on the general suitability of
fragments could bias the behavior of their fusion partners. Split
fluorescent proteins, for example, do not reversibly associate, and Received: September 19, 2015
chromophore maturation tends to be slow, thus obscuring the Accepted: November 16, 2015
dynamics of the target proteins under study.3 β-Galactosidase Published: November 16, 2015

© 2015 American Chemical Society 400 DOI: 10.1021/acschembio.5b00753


ACS Chem. Biol. 2016, 11, 400−408
ACS Chemical Biology Articles

Figure 1. Nluc dissection and optimization. (a) Nluc topology model9 consisting of a 10-stranded beta-barrel. The identified dissection point (red
arrow) occurs between residues 156 and 157 and generates a 156-amino acid polypeptide (156) and a 13-amino acid peptide (native peptide, NP). (b)
Nluc Western blot of HEK293T lysates expressing empty vector (−), 156, 11S, or Nluc. β-actin was included as a loading control. (c) Intracellular half-
life of Nluc, 156, and 11S in HeLa cells following treatment with cycloheximide, as measured by enzyme activity, i.e., normalized relative luminescence
units (RLU; with saturating NP for 156 and 11S). (d) Thermal stability of purified Nluc, 156, and 11S, based on remaining activity at ambient
temperature in the presence of saturating NP added 30 min after temperature challenge. (e) Specific activities of 156 and 11S normalized to Nluc.
Equimolar amounts of each were used to determine Vmax values (under conditions of saturating NP for 156 and 11S). (f) Titration of 11S with various
peptides demonstrating the broad range (>105) of binding affinity. KD values (shown in parentheses) were determined by fitting the data to a one site
specific binding model. For panels c−f: n = 3, variability displayed as SD.

this approach for providing accurate results. Although qualitative designed to be quantifiable within living cells at relevant
correlation may be sufficient for certain experimental purposes, concentrations and temperatures.
accurate representations of intracellular dynamics would We accomplished this through the application of two unique
normally be preferred. design strategies. First, beyond simply identifying an optimal split
NanoLuc (Nluc) is an engineered luciferase derived from a site, we further enhanced efficacy through structural optimization
deep sea luminous shrimp.9 The enzyme is small (19 kDa), of the individual components. Although this approach had
stable, and produces bright and sustained luminescence. Taking already been applied in the development of Nluc,9 we expanded
advantage of these attributes, we sought to create a binary it to address issues specific to a dynamic binary structure. The
reporter system having minimal steric burden on the fusion second strategy resulted from our fortuitous discovery that Nluc
partners but still providing high detection sensitivity. Our could be dissected 13 amino acids from its C-terminus. We
objective was to allow accurate quantitation of protein envisioned that such a small tag would minimize steric conflicts
interactions under physiological conditions relevant to the on fusion partners where geometric constraints may be critical.
cellular environment. Therefore, in addition to having a minimal This design strategy also allowed reporter stability and intrinsic
physical presence, our reporter system was designed to have affinity to be separately engineered. Stability was optimized in the
minimal influence on the affinity and association kinetics of the larger component, which has sufficient size to retain an
interacting target proteins. Furthermore, the reporter was independent tertiary structure. Intrinsic affinity was then
401 DOI: 10.1021/acschembio.5b00753
ACS Chem. Biol. 2016, 11, 400−408
ACS Chemical Biology Articles

Figure 2. Influence of NanoBiT on the SME1/BLIP interaction. (a) Inhibition profiles of the SME1/BLIP WT interaction with differing combinations
of fused NanoBiT. Each profile was normalized by the β-lactamase (SME1) activity measured in the absence of BLIP. (b) Inhibition constants (Ki)
determined by β-lactamase activity with different combinations of fused NanoBiT. Corresponding dissociation constants (KD) determined by luciferase
activity where both SME1 and BLIP were fused to NanoBiT. (c) Relationship between NanoBiT luminescence and inhibition of SME1 (both
normalized). (d) Increase in luminescence with time after the addition of BLIP-114 at various concentrations (0.5−30 nM). (e) Observed rate constants
(kobs) for each concentration of BLIP-114. (f) Association (kon) and dissociation (koff) rate constants of BLIP binding to SME1 calculated from NanoBiT
measurements. KD values calculated from the ratio of koff/kon. For all panels: n = 3, variability displayed as SD.

separately adjusted with the smaller component, which is too Our results reveal that simply splitting a reporter protein may
short to support a tertiary structure on its own. not necessarily provide optimal fragments for quantifying protein
402 DOI: 10.1021/acschembio.5b00753
ACS Chem. Biol. 2016, 11, 400−408
ACS Chemical Biology Articles

interactions. Rather, compromised structural integrity resulting following inhibition of protein synthesis was monitored (Figure
from protein fragmentation can reduce the activity of the s5a,b). No significant effect on protein turnover was evident with
reporter and as a result decrease detection sensitivity. Addition- either 11S or parental 156. A slight increase in degradation may
ally, the intrinsic affinity of the fragments can limit the dynamic be associated with attaching the unstructured NP (or a modified
range of the reporter response and potentially interfere with the variant; Figure s5c−e).
interaction characteristics of the target proteins. By adapting the The apparent KD of 11S with NP was 900 nM (Figure 1f;
structure of these fragments to serve as components of a binary Table s2), indicating that the affinity may be too high for use with
reporter system, we have improved their analytical proficiency weakly interacting proteins. Ideally, the intrinsic affinity of the
for revealing dynamic intracellular processes occurring under complementation reporter should be substantially lower than the
physiological conditions. In experiments designed to evaluate affinity of the interacting target proteins (with affinities in the
quantitative concordance of the luminescent signal with protein high nM to μM range). To reduce the intrinsic affinity of the
interactions, this complementation reporter based on Nluc reporter, a semirandom library of 350 variants of NP comprising
delivered accurate representations of both affinity and generally conservative amino acid substitutions (or deletions at
association kinetics. As the components of this optimized each terminus) was synthesized and screened for affinity and
reporter are structurally distinct from Nluc and for nomenclature luminescence with 11S. This uncovered complementing
simplicity, we refer to the reporter as NanoBiT (shorthand for peptides with affinities that spannned 5 orders of magnitude,
NanoLuc Binary Technology).


yet for many of the peptides the specific activity (i.e., Vmax) across
the variants was relatively unaffected (Figure 1f; Table s2). The
RESULTS AND DISCUSSION lowest affinity was achieved with 114, an 11-amino acid peptide
Engineering a Complementation Reporter. To identify (1.3 kDa) exhibiting an apparent KD of 190 μM. The
potential dissection points in Nluc, we created circularly luminescence spectrum of 11S with 114 is essentially unchanged
permutated10 variants in which the N- and C-termini were from that of Nluc (peak emission ∼460 nm; Figure s6). The
relocated within the protein structure, and the original termini weak association between 11S and 114 indicates that they should
were tethered by a cleavable peptide linker (i.e., containing a not significantly alter the binding affinity of target proteins having
recognition sequence for TEV protease). Ninety variants, KD values up to ∼10 μM.
representing permutation sites distributed throughout the Nluc Further investigation revealed that peptides even shorter than
sequence, were generated and screened in cell lysates for 11 amino acids could deliver productive complementation with
luciferase activity. Measurements were made both before and 11S, although with some loss of light intensity (Table s3; Figure
after proteolytic cleavage to identify fragments that require a s7). In addition, we found that a much shorter linker (Table s4;
proximity constraint for maintaining luciferase activity (Figure Figure s8) or no linker at all (Table s5; Figure s9) may be
s1a). By this method, a dissection point was found between sufficient for the peptide. Having a shorter complementary
residues 156 and 157 (Nluc CP-157; Figure 1a; Figure s1b−g), peptide or linker may be beneficial in circumstances where a
leading to an 18 kDa polypeptide fragment (termed 156) and a minimal fusion tag is preferred.
much smaller 13-amino acid (1.5 kDa) peptide (termed native Accuracy of Interaction Measurements. We used the
peptide, or NP). The binding affinity (KD = 6 μM) between 156 interaction between purified SME-1 β-lactamase (SME1) and β-
and NP is comparable to that between fragments of a commonly lactamase inhibitory protein (BLIP)13 as a model to assess the
used split firefly luciferase (Fluc;11 Figure s2), suggesting their
accuracy of 11S combined with 114 (NanoBiT) for quantifying
potential suitability as a complementation reporter. However,
protein interactions. This model allowed binding interactions to
156 expressed poorly in cells (Figure 1b) and was rapidly
be measured either through inhibition of β-lactamase or by
degraded compared to full-length Nluc (Figure 1c). This was
NanoBiT luminescence. Moreover, using reported affinity
likely a result of compromised structural stability associated with
fragmentation, as evidenced by reduced thermal stability of the mutants of BLIP,14 we could evaluate a range of binding
luminescent activity (Figure 1d) and formation of insoluble interactions without altering the overall molecular geometry.
aggregates in E. coli (Figure s3a). Specifically, we used BLIP WT (Ki = 2.4 nM), BLIP Y50A (Ki =
To improve stability, 156 was randomly mutated and screened 32 nM), and BLIP R160A (Ki = 322 nM) to provide a ∼100-fold
for luminescence in E. coli lysates containing NP. Higher range in binding affinity.
luminescence should result from increased enzyme expression or Evaluation of different linkage orientations revealed optimal
increased specific activity, either of which were expected to performance with 11S appended to the C-terminus of SME1 and
correlate with improvements in structural stability. Two rounds 114 appended to the C-terminus of BLIP. This configuration
of mutagenesis and screening (nearly 15,000 variants) uncovered apparently imposed minimal steric constraints, since appending
16 beneficial amino acid substitutions which were combined to differing permutations of this configuration had little effect on
produce the variant, 11S (Figures s3, s4a; Table s1). In the inhibition profile of SME1 with the various BLIPs (Figure
mammalian cells, 11S exhibited greater protein expression 2a,b; Figure s10a,b). The estimated Ki values corresponded to
(Figure 1b) and reduced protein turnover (Figure 1c) to levels published measurements,14 with small differences between each
comparable with Nluc. Luminescence expression, assayed in the other (2-fold or less) associated mostly with fusions of 114 to the
presence of NP, was also increased by over 300-fold (Figure s4b), BLIPs. From the configuration containing both subunits of
with specific activity increased 3-fold (Figure 1e) to 37% of Nluc. NanoBiT, the measurements of luminescence corresponded
Thermal stability of the luminescence (i.e., Tm) increased from closely with the inhibition of β-lactamase activity (Figure 2c;
48 to 54 °C (Figure 1d). Figure s10c,d). Values for Ki (β-lactamase inhibition) and KD
While appending an unstable polypeptide could potentially (NanoBiT bioluminescence) were essentially indistinguishable
promote degradation of a target protein, it could also result in within the precision of the assays (Figure 2b), indicating that
suppression of degradation. To investigate this, 11S and 156 were NanoBiT provided an accurate assessment of protein inter-
fused to Fluc-PEST (FlucP),9,12 and the loss of FlucP activity actions in this model system.
403 DOI: 10.1021/acschembio.5b00753
ACS Chem. Biol. 2016, 11, 400−408
ACS Chemical Biology Articles

To assess the ability to quantify interaction kinetics, we also 99%), consistent with essentially complete dissociation of FRB/
used NanoBiT to estimate the association (kon) and dissociation FKBP. As expected, the interaction profile in cell lysates shifts
(koff) rate constants for SME1 with the BLIPs. In contrast to the strongly to the right due to dilution of interacting proteins into
assay for β-lactamase (colorimetric conversion of nitrocefin), the the culture medium. The lower baseline results from reduced
instantaneous production of luminescence allows dynamic spontaneous luminescence (i.e., autoluminescence) generated by
processes to be continuously monitored. The increase in furimazine in the lytic reagent.
luminescence upon combining BLIP with SME1 conformed Intracellular Kinetics. As cellular processes are generally
well to a pseudo-first-order reaction (Figure 2d; Figure s11a,b), reactive to environmental conditions, it is advantageous for the
and kobs values correlated linearly with BLIP concentrations reporter to also indicate the dynamic character of protein
(Figure 2e; Figure s10c,d). The data indicate that reduced affinity interactions. This is exemplified by the transent interaction of β-
of the BLIP mutants was achieved largely by increased koff, with arrestin-2 (ARRB2) with the β2-adrenergic receptor (ADRB2),
relatively little effect on kon. Values of KD calculated by the ratio of in contrast to the more stable interaction it makes with the
koff to kon corresponded well to the estimates of KD made under arginine vasopressin receptor 2 (AVPR2).15 This kinetic
steady-state conditions (Figure 2f). distinction was apparent by expressing ADRB2-11S/114-
The intrinsic kon and koff rate constants for NanoBiT were ARRB2 or AVPR2-114/11S-ARRB2 in HEK293T cells treated
determined to be ∼500 M−1 s−1 and 0.2 s−1, respectively (Figure with the appropriate agonist (isoproterenol and vasopressin,
s12). The BLIP having the lowest affinity (R160A, measured KD respectively; Figure 4a). Temporal differences were also
∼ 1 μM) also had the lowest kon and highest koff values. As the kon observed by bioluminescence imaging of HeLa cells expressing
for NanoBiT was ∼10-fold lower than for R160A and the koff ∼ the NanoBiT fusions (Figure s14a−d).
10-fold higher, the intrinsic kinetics of NanoBiT should not affect More kinetically challenging is the interaction of protein
the interaction kinetics of R160A with SME1. The effect of kinase A catalytic (PRKACA) and type 2A regulatory
NanoBiT should be even less for the higher affinity BLIPs. (PRKAR2A) subunits in response to intracellular cAMP
NanoBiT may begin to influence the apparent kinetics of protein concentrations. Previous studies have shown that dynamic
interactions having unusually slow association rates or fast changes in the association of these subunits can occur rapidly in
dissociation rates, which may underly very weak binding affinities response to applying external stimuli to the cells.15 We explored
(e.g., KD > 10 μM). the ability of NanoBiT to represent these rapid dynamics by
Protein Interactions in Cells. For NanoBiT to produce expressing 114-PRKACA and 11S-PRKAR2A in HEK293T cells,
quantitative data in living cells, its luminescence must be linear along with a luminescence-based biosensor for intracellular
with the intracellular concentration of the binary complex. cAMP.16 By utilizing a different substrate (Fluc luciferin), the
Linearity of Nluc luminescence with reporter concentration has biosensor can be independently assayed within cells. Modulation
previously been established biochemically.9 In both live cells and of the endogenous ADRB2 receptor through progressive
cell lysates, luminescence was also proportional to the amount of addition of an agonist (isoproterenol) and antagonist (propra-
Nluc DNA used for transfection (Figure 3), although deviating nolol), followed by stimulation of adenylate cyclase with
forskolin, caused rapid changes in cAMP concentrations as
indicated by the biosensor. Corresponding inverse changes were
evident in NanoBiT luminescence, showing precise temporal
coupling of the reporters in response to the underlying cellular
dynamics (Figure 4b).
As molecular kinetics are fundamentally linked to temperature,
measurements at the relevant physiological temperature are
required to accurately represent interaction dynamics. The
response profile of protein kinase A represented by NanoBiT
showed that both association and dissociation rates were much
faster at 37 °C than 21 °C (i.e., ambient temperature; Figure
5a,b). The analogous experiment using split Fluc as the
Figure 3. Linearity of luminescence in cells and cell lysates. HEK293T complementation reporter (i.e., PRKACA-FlucC/FlucN-
cells transfected with NanoBiT (equal amounts of FRB-11S and FKBP- PRKAR2A) showed a marked deterioration of the data quality
114) or Nluc DNA were treated (±) with 30 nM rapamycin (R) and at physiological temperature. This is consistent with a previous
measured for luminescence before or after cell lysis. n = 3 (NanoBiT) or report describing the unstable nature of split Fluc.17 Because the
n = 6 (Nluc), variability displayed as SD. Curve fits for Nluc and 114 peptide is unlikely to have conformationally defined
NanoBiT were generated based on the expected first order and second structure in the absence of 11S, the suitable performance of
order relationships, respectively. NanoBiT at higher temperature was likely due to the thermal
stability of the 11S subunit.
slightly at high expression. To assess linearity for NanoBiT, the Pharmacological Modulation of Protein Interactions.
rapamycin-inducible FRB/FKBP model was used (i.e, FRB-11S/ Because complementation reporters are frequently used to
FKBP-114; Figure s13a,b). At higher DNA concentrations, the investigate the influence of synthetic molecules on protein
intracellular luminescence from NanoBiT in the presence of interactions, we evaluated the suitability of NanoBiT for this type
rapamycin followed a linear trend similar to Nluc. The signal of application. The effect of intrinsic affinity between the
departed significantly at lower concentrations, suggesting some complementation subunits was investigated by expressing FRB-
dissociation of FRB/FKBP even in the presence of rapamycin. 11S in HEK293T cells, together with FKBP fused to peptides
These data conform to a second order model for the with differing affinities to 11S. Our results indicate that the
concentration-dependent interaction of two proteins. Lumines- apparent potency of rapamycin to induce protein dimerization
cence was greatly reduced in the absence of rapamycin (i.e., > was minimally affected by the intrinsic affinity (range >100,000
404 DOI: 10.1021/acschembio.5b00753
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ACS Chemical Biology Articles

Figure 4. Monitoring temporal interactions in cells. (a) ARRB2 interaction with fast or slowly recycling GPCRs. The interactions of ADRB2-11S/114-
ARRB2 and AVPR2-114/11S-ARRB2 were followed continuously in HEK293T cells at 37 °C after treatment at t = 1 min with isoproterenol (10 μM) or
Arg8-vasopressin (100 nM), respectively. Individual S/B values were scaled by their respective dynamic ranges by subtracting the minimum value of the
data set, then dividing by the maximum value. (b) Reversible interaction of 114-PRKACA/11S-PRKR2A followed continuously in HEK293T cells at 28
°C. 10 μM isoproterenol (Iso), propranolol (Pro), and forskolin (Fsk) were added sequentially to modulate endogenous ADRB2 (Iso and Pro) or
activate endogenous adenylate cyclase (Fsk). Changes in intracellular cAMP concentration were monitored independently using GloSensor cAMP.
Data were scaled by respective dynamic ranges as in panel a. For both panels: n = 3, variability displayed as SD.

Figure 5. Influence of temperature on monitoring PKA. Comparison of NanoBiT and split Fluc for monitoring PKA interaction dynamics at 21 °C (a)
and 37 °C (b). HEK293T cells transiently expressing PRKACA and PRKAR2A fused to NanoBiT (50 pg DNA/well) or split Fluc (5 ng DNA/well)
were treated as described in Figure 4b. Data were normalized as described for Figure 4b. For both panels: n = 3, variability displayed as SD. Data for other
DNA concentrations can be found in Figure s15.

examined; Figure 6a; Figure s16a−i), and the values achieved are pathway in cells expressing wild type BRAF.19 This phenomenon
consistent with published reports.18 This should be expected has been monitored in cells previously using kinase domains
since compound potency is typically associated with binding modified with the CAAX membrane targeting motif.20 We
affinity to its target, rather than interaction affinity within the surmised that the small size and stability of NanoBiT may allow
target complex. Thus, potency should be minimally influenced by drug-induced dimerization to be detected in the full-length
the choice of complementation reporter so long as the binding proteins. In cells expressing BRAF-11S/CRAF-114, an inhibitor-
site for the compound is unaltered. In contrast, the dynamic dependent increase in dimerization was observed, with the
response induced by rapamycin is strongly dependent on the calculated EC50 for each inhibitor in agreement with previously
intrinsic affinity of the reporter (Figure 6b). This arises primarily described potencies20 (Figure 7a). Cells expressing the NanoBiT
from increased luminescence of the sample lacking rapamycin, fusions exhibited increased phosphorylation of MEK and ERK in
presumably due to increased spontaneous interaction promoted correlation with drug-induced dimerization21 (Figure s18a,b).
by the interaction affinity of the reporter subunits. Thus, the NanoBiT was also capable of differentiating between the
highest assay sensitivity was achieved with the low affinity 114 reversible inhibitor, GDC 0879, and the irreversible inhibitor, AZ
peptide (S/B = 1,200; Z′-factor =0.98). A similar analysis was 628. Removal of GDC 0879 from the cells caused the
performed with split Fluc (Figure s17). luminescence to decrease, indicating dissociation of BRAF/
More pharmaceutically relevant is the interaction of BRAF CRAF. In contrast, removal of AZ 628 had no effect, indicating
with CRAF, which paradoxically is induced by inhibitors that it remained bound to its target (Figure 7b). These results
designed for oncogenic mutants of BRAF. Although intended indicate that the intrinsic affinity of the NanoBiT subunits is
to promote tumor regression by suppressing the RAS-RAF- suitable for quantification of both the potency and kinetics of
MEK-ERK signaling pathway, these inhibitors can activate the drug-induced protein interactions.
405 DOI: 10.1021/acschembio.5b00753
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ACS Chemical Biology Articles

Figure 6. Influence of NanoBiT on rapamycin-inducible FRB/FKBP interaction in cells. (a) Rapamycin potency (EC50) for inducing the interaction
between FRB-11S and FKBP fused to various peptides. (b) Induction of protein interactions with 30 nM rapamycin in HEK293T cells coexpressing
FRB-11S and FKBP-peptide. For both panels: n = 4, variability displayed as SD.

conformational stability and low intrinsic affinity. Increased


stability designed into the large component (11S) provides
improved expression and reporter performance at physiological
temperature. Low intrinsic affinity, achieved by adapting the
small peptide component (114), minimizes its influence on the
interaction characteristics of the target proteins. The intrinsic
affinity and association constants determined for NanoBiT were
found to be outside of the ranges typical for protein interactions.
Thus, the luminescent signal from NanoBiT should provide an
accurate indication of interaction dynamics (e.g., for proteins
having KD < 10 μM), although caution is prudent when working
with unusually weak or transient interactions.
This capability was verified using SME1 in combination with a
set of interacting inhibitory proteins (BLIP) of differing affinities.
Appending the reporter had negligible influence on binding
affinity, although as with any fusion tag, the possibility of steric
interference for certain protein pairs should be considered.
Nonetheless, the luminescent signal from NanoBiT correlated
precisely with the independent assessment of protein association
through SME1 activity. In mammalian cells, the luminescence
produced by NanoBiT is well behaved and reflective of the
interaction status of the reporter. For the interaction of FKB/
Figure 7. Measuring inhibitor-induced BRAF/CRAF dimerization. (a) FRBP, it showed second-order behavior consistent with a
BRAF-11S and CRAF-114 dimerization in HCT116 cells following 2 h dynamic binary system, while at higher concentrations favoring
incubation with different BRAF inhibitors. Calculated EC50 values full association of the dimeric complex, it conformed to the linear
shown in legend. (b) Washout experiment showing reversibility of characteristics of NanoLuc. The luminescent response was rapid
BRAF-11S/CRAF-114 interaction. HEK293T cells were treated with 1
μM of the reversible inhibitor, GDC 0879, or the covalent inhibitor, AZ and reversible without evident lag, indicating that NanoBiT can
628, for 2 h. For washout samples, inhibitor was removed by media react nearly instantaneously to changing intracellular conditions.
exchange and luminescence was measured 1 h later. Data represent the Furthermore, dynamic processes can be monitored for more
remaining luminescent signal normalized to t = 0 of the curve fit. For than an hour due to the stable signal duration of the luminescent
both panels: n = 4, variability displayed as SD. reaction.
Our analysis indicates that NanoBiT fulfills the general
Summary. NanoBiT is a complementation reporter designed expectation of a complementation reporter in mammalian cells,
for quantitative investigation of protein interaction dynamics with biomolecular parameters generally suited for accurate
under relevant physiological conditions. Benefiting from the portrayal of intracellular processes. In addition to analyzing
small size and bright luminescence of NanoLuc, it provides protein interactions within cells, we also confirmed the ability to
detection at low intracellular concentrations with minimal steric
characterize pharmacological outcomes. Specifically, NanoBiT
interference on appended target proteins. Notably, one of the
NanoBiT componenents is only 11 amino acids long, can reveal drug potency for induced protein interactions and the
comparable to other peptide tags routinely used in protein binding kinetics underlying drug action. Moreover, with the
analysis. The other NanoBIT component is also relatively small ability to substantially alter both size and intrinsic affinity of the
in terms of protein tags (e.g., two-thirds the size GFP). peptide component in this binary reporter, we believe this
NanoBiT is distinct from other complementation reporters luminescent system will provide additional applications yet to be
because its components are structurally optimized for high realized.
406 DOI: 10.1021/acschembio.5b00753
ACS Chem. Biol. 2016, 11, 400−408
ACS Chemical Biology Articles

■ METHODS
DNA Constructs, Protein Purification, and Lysate Prepara-
RLUmax =
Vmax × [Fz]
KM + [Fz].
tion. See the Supporting Information.
Nluc Dissection and Screening. CP Nluc variants were made Peptide Affinities. Purified 11S (40 pM) or 156 (400 pM) were
using splicing by overlap extension (SOE).22 Each variant was designed mixed with various concentrations of synthetic peptide (Peptide 2.0) in
so that former termini were joined by a 33-amino acid polypeptide linker assay buffer and incubated at ambient temperature for 30 min. Nano-
containing a TEV protease sequence (underlined): Glo Luciferase Assay Reagent was added and luminescence measured on
GSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGG. Variants were a GloMax-Multi+ Detection System. Average RLUs over 10 min were fit
screened by incubating bacterial, wheat germ, or mammalian lysates (one site specific binding) using GraphPad Prism 6, and the fits were
with or without ProTEV Plus in 1X ProTEV buffer (Promega) at 30 °C subsequently used to determine KD values.
for 60 min. Samples were diluted in DMEM and then further diluted 1:2 β-Lactamase Assays. Purified BLIP variants were incubated with
in Nano-Glo Luciferase Assay Reagent (Promega) for 5 min at ambient SME1 for 2 h at ambient temperature. Nitrocefin (EMD Millipore) was
temperature before measuring luminescence with a GloMax-Multi+ added to the equilibrated complex, and substrate hydrolysis was
Detection System (Promega). monitored (A280) every 25 s for 25 min on an Infinite M1000 reader
Sequence Optimization. DNA encoding Nluc amino acids 1−156 (Tecan). Using these measurements, inhibition constants were
was mutated by error-prone PCR (Diversify PCR Random Mutagenesis, calculated as previously described.13
Clontech), subcloned into pF4Ag,9 and used to transform KRX E. coli. Luminescence-based Binding Assays. KD values were deter-
Lysates of 3,696 variant clones were screened (Freedom robotic mined by adding Fz (10 μM) to the equilibrated complex, measuring
workstation (Tecan)) by incubation with HaloTag-NP (KRX lysates) luminescence on a GloMax-Multi+ Detection System every 1 min for 30
for 10 min with shaking. Nano-Glo Luciferase Assay Reagent was added, min, and fitting the data to the equation,
and luminescence was measured on a GENiosPro reader (Tecan). In
addition to fully random mutagenesis, we attempted to engineer RLUmax × [BLIP]
RLU =
structural stability to 156 by reducing conformational entropy.23 Each KD + [BLIP].
glycine residue was substituted to alanine, and beneficial (∼1.5-fold
improved luminescence) changes (G15A, G35A, G51A, G67A, and For BLIP binding rate constants, BLIP-114, SME1-11S, and Fz were
G71A) were combined with mutations identified from the random combined, and luminescence was monitored for 15 min (BLIP WT-
library screen. The combined variant providing the highest 114) using a GloMax-Multi+ Detection Systemor every 1 s for 100 s
luminescence was used as the template for a second library, where (BLIP Y50A-114 and BLIP R160A-114) using the Infinite M1000
11,088 variants were screened in similar fashion. Improved variants reader (Tecan). Observed rate constants (kobs) were determined by
identified during primary screening were validated using a secondary fitting (one phase association) with GraphPad Prism 6. The observed
screen, where clones were processed the same way but in triplicate. rate constant was then plotted versus BLIP-114 concentration, and the
Verified hits were then sequenced. linear fit was determined. The association and dissociation rate
Synthetic peptide arrays (≥75% purity; N-terminus acetylated and constants were determined by fitting kobs data to the equation,
the C-terminus amidated; New England Peptide) were designed to
semirandomly sample combinatorial sequence space (via generally kobs = kon × [BLIP] + koff .
conservative amino acid substitutions and deletions at each terminus)
across NP. Peptides were dissolved in water and concentration Intracellular Luminescence. For each model, the eight possible
quantified by A280. All other synthetic peptides of interest were orientations of the reporter subunits fused onto the interacting proteins
synthesized at >95% purity (Peptide 2.0). Relative binding affinity and were screened for maximal S/B. Using the optimal orientations,
maximal luminescence were determined by titrating the peptide into mammalian cells transiently expressing the fusion proteins (1:1 ratio of
large NanoBiT variants as described below under peptide affinities. interacting pairs) were plated at 2 × 104 cells/well (96-well plate,
Intracellular and Thermal Stability. HeLa cells expressing 156, Corning) and incubated ± drug at 37 °C. For FRB/FKBP, HEK293T
11S, or Nluc were plated at 104 cells/well (96-well plate, Corning), and cells were incubated for 2 h with rapamycin (EMD Millipore) in Opti-
24 h later medium was exchanged with growth medium containing 0.4 MEMI. For BRAF/CRAF, HEK293T or HCT116 cells were serum
mM cycloheximide or DMSO. Cells were assayed over 6 h by addition of starved for 4 h in Opti-MEMI before incubation for 1 h with GDC 0879
Nano-Glo Luciferase Assay Reagent (with 100 μM NP for 156 and 11S) (R&D Systems), AZ 628 (R&D Systems), Sorafenib (Cell Signaling
and measured on a GloMax-Multi+ Detection System. Cycloheximide- Technology), or PLX4720 (Cayman Chemical Company). Nano-Glo
treated samples were normalized to DMSO-treated samples for each Live Cell Substrate (Promega) was added to a 1× concentration, and
time point, and data were fit (one phase decay) using GraphPad Prism 6. luminescence was measured using a GloMax-Multi+ Detection System.
Thermal stability analysis was carried out by incubating 1.5 pmol 156 For kinetic measurements, HEK293T cells were plated at 1 × 104
or 30 fmol 11S (in DMEM containing 0.1% Prionex) for 30 min at cells/well and transfected the following day with ADRB2, AVPR2,
different temperatures. Following rapid cooling to 4 °C, 300 pmol of ARRB2, PRKAR2A, or PRKACA DNA. Twenty-four hours post-
synthetic NP was added, and samples were incubated at 22 °C for 5 min. transfection, growth medium was exchanged with Opti-MEMI, and cells
Nano-Glo Luciferase Assay Reagent was added, and luminescence was were incubated for 3.5 h at 37 °C. Nano-Glo Live Cell Substrate was
measured using an Infinite F500 reader (Tecan). Activity was added, and luminescence was measured using a Varioskan Flash
normalized to control samples incubated at 4 °C. Tm represents the Multimode Reader (Thermo Fisher Scientific) at either 21, 28, or 37 °C.
temperature at which 50% of protein is inactive. Following thermal equilibration, compound was added by injection to
Specific Activity. Purified Nluc, 156, and 11S were diluted to 40 pM final concentrations of 10 μM (isoproterenol, propranolol, forskolin) or
in assay buffer (PBS pH 7, 0.01% Prionex, 0.005% Tergitol, 1 mM 100 nM (Arg8-vasopressin) in Opti-MEMI and measurements
DTT). Synthetic NP was titrated (64 nM−140 μM for 156; 9 nM−20 continued. Split Fluc assays were performed in the same manner except
μM for 11S; buffer in place of peptide for Nluc) and samples assayed using 2 mM Luciferin-EF (Promega). The GloSensor cAMP Assay
with variable furimazine (Fz) concentrations (78 nM−10 μM for Nluc; (Promega) was used to monitor the cAMP response following the
625 nM−20 μM for 156/11S) in Nano-Glo Buffer. For each manufacturer’s recommended protocol at variable temperatures and
concentration, maximal luminescence (RLUmax) was calculated using with the addition of 2% (v/v) GloSensor cAMP Reagent 30 min prior to
the equation, beginning luminescence measurements.
Curve fitting luminescence linearity in cells and lysates was modeled
RLUmax × [Peptide] using the expected first order relationship between Nluc expression and
RLU =
KD + [Peptide]. RLUs (a[DNA]+b) and the expected second order relationship for
NanoBiT (a[DNA]2/(k+[DNA])+b).
Vmax was then calculated using the equation, Additional methods can be found in the Supporting Information.

407 DOI: 10.1021/acschembio.5b00753


ACS Chem. Biol. 2016, 11, 400−408
ACS Chemical Biology Articles


*
ASSOCIATED CONTENT
S Supporting Information
(11) Paulmurugan, R., and Gambhir, S. S. (2007) Combinatorial
library screening for developing an improved split-firefly luciferase
fragment-assisted complementation system for studying protein-protein
The Supporting Information is available free of charge on the interactions. Anal. Chem. 79, 2346−2353.
ACS Publications website at DOI: 10.1021/acschem- (12) Swanson, B., Fan, F., and Wood, K. V. (2007) Enhanced response
bio.5b00753. dynamics for transcription analysis using new pGL4 luciferase reporter
Figures s1−s18, Tables s1−s5, and supporting methods vectors. Cell Notes 17, 3−5.
(13) Zhang, Z., and Palzkill, T. (2003) Determinants of binding affinity
(PDF) and specificity for the interaction of TEM-1 and SME-1 beta-lactamase


with beta-lactamase inhibitory protein. J. Biol. Chem. 278, 45706−45712.
AUTHOR INFORMATION (14) Petrosino, J., Rudgers, G., Gilbert, H., and Palzkill, T. (1999)
Contributions of aspartate 49 and phenylalanine 142 residues of a tight
Corresponding Author binding inhibitory protein of beta-lactamases. J. Biol. Chem. 274, 2394−
*Tel.: 608-274-1181. E-mail: lance.encell@promega.com. 2400.
Present Address (15) Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron,
§ M. G. (1999) Association of beta-arrestin with G protein-coupled
Department of Pharmaceutics and Pharmaceutical Chemistry,
receptors during clathrin-mediated endocytosis dictates the profile of
University of Utah, Salt Lake City, Utah 84112, United States
receptor resensitization. J. Biol. Chem. 274, 32248−32257.
Notes (16) Binkowski, B. F., Butler, B. L., Stecha, P. F., Eggers, C. T., Otto, P.,
The authors declare no competing financial interest. Zimmerman, K., Vidugiris, G., Wood, M. G., Encell, L. P., Fan, F., and


Wood, K. V. (2011) A luminescent biosensor with increased dynamic
ACKNOWLEDGMENTS range for intracellular cAMP. ACS Chem. Biol. 6, 1193−1197.
(17) Ohmuro-Matsuyama, Y., Chung, C. I., and Ueda, H. (2013)
We thank G. Vidugiris for help with screening sequence libraries Demonstration of protein-fragment complementation assay using
and G. Colwell at Gene Dynamics, LLC, for assistance with purified firefly luciferase fragments. BMC Biotechnol. 13, 31.
vector constructions. (18) Banaszynski, L. A., Liu, C. W., and Wandless, T. J. (2005)


Characterization of the FKBP-rapamycin-FRB ternary complex. J. Am.
REFERENCES Chem. Soc. 127, 4715−4721.
(19) Holderfield, M., Nagel, T. E., and Stuart, D. D. (2014) Mechanism
(1) Braun, P. (2012) Interactome mapping for analysis of complex and consequences of RAF kinase activation by small-molecule
phenotypes: insights from benchmarking binary interaction assays. inhibitors. Br. J. Cancer 111, 640−645.
Proteomics 12, 1499−1518. (20) Lavoie, H., Thevakumaran, N., Gavory, G., Li, J. J., Padeganeh, A.,
(2) Michnick, S. W., Ear, P. H., Manderson, E. N., Remy, I., and Stefan, Guiral, S., Duchaine, J., Mao, D. Y., Bouvier, M., Sicheri, F., and
E. (2007) Universal strategies in research and drug discovery based on Therrien, M. (2013) Inhibitors that stabilize a closed RAF kinase
protein-fragment complementation assays. Nat. Rev. Drug Discovery 6, domain conformation induce dimerization. Nat. Chem. Biol. 9, 428−436.
569−582. (21) Hatzivassiliou, G., Song, K., Yen, I., Brandhuber, B. J., Anderson,
(3) Magliery, T. J., Wilson, C. G., Pan, W., Mishler, D., Ghosh, I., D. J., Alvarado, R., Ludlam, M. J., Stokoe, D., Gloor, S. L., Vigers, G.,
Hamilton, A. D., and Regan, L. (2005) Detecting protein-protein Morales, T., Aliagas, I., Liu, B., Sideris, S., Hoeflich, K. P., Jaiswal, B. S.,
interactions with a green fluorescent protein fragment reassembly trap: Seshagiri, S., Koeppen, H., Belvin, M., Friedman, L. S., and Malek, S.
scope and mechanism. J. Am. Chem. Soc. 127, 146−157. (2010) RAF inhibitors prime wild-type RAF to activate the MAPK
(4) Rossi, F., Charlton, C. A., and Blau, H. M. (1997) Monitoring pathway and enhance growth. Nature 464, 431−435.
protein-protein interactions in intact eukaryotic cells by beta- (22) Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L.
galactosidase complementation. Proc. Natl. Acad. Sci. U. S. A. 94, R. (1989) Engineering hybrid genes without the use of restriction
8405−8410. enzymes: gene splicing by overlap extension. Gene 77, 61−68.
(5) Stefan, E., Aquin, S., Berger, N., Landry, C. R., Nyfeler, B., Bouvier, (23) Scott, K. A., Alonso, D. O., Sato, S., Fersht, A. R., and Daggett, V.
M., and Michnick, S. W. (2007) Quantification of dynamic protein (2007) Conformational entropy of alanine versus glycine in protein
complexes using Renilla luciferase fragment complementation applied denatured states. Proc. Natl. Acad. Sci. U. S. A. 104, 2661−2666.
to protein kinase A activities in vivo. Proc. Natl. Acad. Sci. U. S. A. 104,
16916−16921.
(6) Misawa, N., Kafi, A. K., Hattori, M., Miura, K., Masuda, K., and
Ozawa, T. (2010) Rapid and high-sensitivity cell-based assays of
protein-protein interactions using split click beetle luciferase com-
plementation: an approach to the study of G-protein-coupled receptors.
Anal. Chem. 82, 2552−2560.
(7) Remy, I., and Michnick, S. W. (2006) A highly sensitive protein-
protein interaction assay based on Gaussia luciferase. Nat. Methods 3,
977−979.
(8) Takakura, H., Hattori, M., Takeuchi, M., and Ozawa, T. (2012)
Visualization and quantitative analysis of G protein-coupled receptor-
beta-arrestin interaction in single cells and specific organs of living mice
using split luciferase complementation. ACS Chem. Biol. 7, 901−910.
(9) Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L.,
Wood, M. G., Otto, P., Zimmerman, K., Vidugiris, G., Machleidt, T.,
Robers, M. B., Benink, H. A., Eggers, C. T., Slater, M. R., Meisenheimer,
P. L., Klaubert, D. H., Fan, F., Encell, L. P., and Wood, K. V. (2012)
Engineered luciferase reporter from a deep sea shrimp utilizing a novel
imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848−1857.
(10) Yu, Y., and Lutz, S. (2011) Circular permutation: a different way
to engineer enzyme structure and function. Trends Biotechnol. 29, 18−
25.

408 DOI: 10.1021/acschembio.5b00753


ACS Chem. Biol. 2016, 11, 400−408