4 research outputs found
A Bidirectional System for the Dynamic Small Molecule Control of Intracellular Fusion Proteins
Small
molecule control of intracellular protein levels allows temporal
and dose-dependent regulation of protein function. Recently, we developed
a method to degrade proteins fused to a mutant dehalogenase (HaloTag2)
using small molecule hydrophobic tags (HyTs). Here, we introduce a
complementary method to stabilize the same HaloTag2 fusion proteins,
resulting in a unified system allowing bidirectional control of cellular
protein levels in a temporal and dose-dependent manner. From a small
molecule screen, we identified <i>N</i>-(3,5-dichloro-2-ethoxybenzyl)-2<i>H</i>-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1)
that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2
ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS
system in probing the physiological role of therapeutic targets by
modulating HaloTag2-fused oncogenic H-Ras, which resulted in either
the cessation (HyT) or acceleration (HALTS) of cellular transformation.
In sum, we present a general platform to study protein function, whereby
any protein of interest fused to HaloTag2 can be either degraded 10-fold
or stabilized 5-fold using two corresponding compounds
Computational Design of Enone-Binding Proteins with Catalytic Activity for the MoritaāBaylisāHillman Reaction
The MoritaāBaylisāHillman reaction forms
a carbonācarbon
bond between the Ī±-carbon of a conjugated carbonyl compound
and a carbon electrophile. The reaction mechanism involves Michael
addition of a nucleophile catalyst at the carbonyl Ī²-carbon,
followed by bond formation with the electrophile and catalyst disassociation
to release the product. We used Rosetta to design 48 proteins containing
active sites predicted to carry out this mechanism, of which two show
catalytic activity by mass spectrometry (MS). Substrate labeling measured
by MS and site-directed mutagenesis experiments show that the designed
active-site residues are responsible for activity, although rate acceleration
over background is modest. To characterize the designed proteins,
we developed a fluorescence-based screen for intermediate formation
in cell lysates, carried out microsecond molecular dynamics simulations,
and solved X-ray crystal structures. These data indicate a partially
formed active site and suggest several clear avenues for designing
more active catalysts
Structural and Biochemical Characterization of the Bilin Lyase CpcS from Thermosynechococcus elongatus
Cyanobacterial phycobiliproteins
have evolved to capture light
energy over most of the visible spectrum due to their bilin chromophores,
which are linear tetrapyrroles that have been covalently attached
by enzymes called bilin lyases. We report here the crystal structure
of a bilin lyase of the CpcS family from Thermosynechococcus
elongatus (<i>Te</i>CpcS-III). <i>Te</i>CpcS-III is a 10-stranded Ī² barrel with two alpha helices and
belongs to the lipocalin structural family. <i>Te</i>CpcS-III
catalyzes both cognate as well as noncognate bilin attachment to a
variety of phycobiliprotein subunits. <i>Te</i>CpcS-III
ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to
the alpha and beta subunits of allophycocyanin and to the beta subunit
of phycocyanin at the Cys82-equivalent position in all cases. The
active form of <i>Te</i>CpcS-III is a dimer, which is consistent
with the structure observed in the crystal. With the use of the UnaG
protein and its association with bilirubin as a guide, a model for
the association between the native substrate, phycocyanobilin, and <i>Te</i>CpcS was produced
Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis
Nucleophilic catalysis is a general strategy for accelerating
ester
and amide hydrolysis. In natural active sites, nucleophilic elements
such as catalytic dyads and triads are usually paired with oxyanion
holes for substrate activation, but it is difficult to parse out the
independent contributions of these elements or to understand how they
emerged in the course of evolution. Here we explore the minimal requirements
for esterase activity by computationally designing artificial catalysts
using catalytic dyads and oxyanion holes. We found much higher success
rates using designed oxyanion holes formed by backbone NH groups rather
than by side chains or bridging water molecules and obtained four
active designs in different scaffolds by combining this motif with
a Cys-His dyad. Following active site optimization, the most active
of the variants exhibited a catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub>) of 400 M<sup>ā1</sup> s<sup>ā1</sup> for the cleavage of a <i>p</i>-nitrophenyl
ester. Kinetic experiments indicate that the active site cysteines
are rapidly acylated as programmed by design, but the subsequent slow
hydrolysis of the acyl-enzyme intermediate limits overall catalytic
efficiency. Moreover, the Cys-His dyads are not properly formed in
crystal structures of the designed enzymes. These results highlight
the challenges that computational design must overcome to achieve
high levels of activity