4 research outputs found
Mapping the H‑NOX/HK Binding Interface in <i>Vibrio cholerae</i> by Hydrogen/Deuterium Exchange Mass Spectrometry
Heme-nitric
oxide/oxygen binding (H-NOX) proteins are a group of hemoproteins
that bind diatomic gas ligands such as nitric oxide (NO) and oxygen
(O<sub>2</sub>). H-NOX proteins typically regulate histidine kinases
(HK) located within the same operon. It has been reported that NO-bound
H-NOXs inhibit cognate histidine kinase autophosphorylation in bacterial
H-NOX/HK complexes; however, a detailed mechanism of NO-mediated regulation
of the H-NOX/HK activity remains unknown. In this study, the binding
interface of <i>Vibrio cholerae</i> (<i>Vc</i>) H-NOX/HK complex was characterized by hydrogen/deuterium exchange
mass spectrometry (HDX-MS) and further validated by mutagenesis, leading
to a new model for NO-dependent kinase inhibition. A conformational
change in <i>Vc</i> H-NOX introduced by NO generates a new
kinase-binding interface, thus locking the kinase in an inhibitory
conformation
Native Alanine Substitution in the Glycine Hinge Modulates Conformational Flexibility of Heme Nitric Oxide/Oxygen (H-NOX) Sensing Proteins
Heme nitric oxide/oxygen
sensing (H-NOX) domains are direct NO
sensors that regulate a variety of biological functions in both bacteria
and eukaryotes. Previous work on H-NOX proteins has shown that upon
NO binding, a conformational change occurs along two glycine residues
on adjacent helices (termed the glycine hinge). Despite the apparent
importance of the glycine hinge, it is not fully conserved in all
H-NOX domains. Several H-NOX sensors from the family Flavobacteriaceae
contain a native alanine substitution in one of the hinge residues.
In this work, the effect of the increased steric bulk within the Ala-Gly
hinge on H-NOX function was investigated. The hinge in <i>Kordia
algicida</i> OT-1 (<i>Ka</i> H-NOX) is composed of
A71 and G145. Ligand-binding properties and signaling function for
this H-NOX were characterized. The variant A71G was designed to convert
the hinge region of <i>Ka</i> H-NOX to the typical Gly-Gly
motif. In activity assays with its cognate histidine kinase (HnoK),
the wild type displayed increased signal specificity compared to A71G.
Increasing titrations of unliganded A71G gradually inhibits HnoK autophosphorylation,
while increasing titrations of unliganded wild type H-NOX does not
inhibit HnoK. Crystal structures of both wild type and A71G <i>Ka</i> H-NOX were solved to 1.9 and 1.6 Ă…, respectively.
Regions of H-NOX domains previously identified as involved in protein–protein
interactions with HnoK display significantly higher b-factors in A71G
compared to wild-type H-NOX. Both biochemical and structural data
indicate that the hinge region controls overall conformational flexibility
of the H-NOX, affecting NO complex formation and regulation of its
HnoK
Regulating the ARNT/TACC3 Axis: Multiple Approaches to Manipulating Protein/Protein Interactions with Small Molecules
For several well-documented reasons, it has been challenging
to develop artificial small molecule inhibitors of protein/protein
complexes. Such reagents are of particular interest for transcription
factor complexes given links between their misregulation and disease.
Here we report parallel approaches to identify regulators of a hypoxia
signaling transcription factor complex, involving the ARNT subunit
of the HIF (Hypoxia Inducible Factor) activator and the TACC3 (Transforming
Acidic Coiled Coil Containing Protein 3) coactivator. In one route,
we used <i>in vitro</i> NMR and biochemical screening to
identify small molecules that selectively bind within the ARNT PAS
(Per-ARNT-Sim) domain that recruits TACC3, identifying KG-548 as an
ARNT/TACC3 disruptor. A parallel, cell-based screening approach previously
implicated the small molecule KHS101 as an inhibitor of TACC3 signaling.
Here, we show that KHS101 works indirectly on HIF complex formation
by destabilizing both TACC3 and the HIF component HIF-1α. Overall,
our data identify small molecule regulators for this important complex
and highlight the utility of pursuing parallel strategies to develop
protein/protein inhibitors
Regulating the ARNT/TACC3 Axis: Multiple Approaches to Manipulating Protein/Protein Interactions with Small Molecules
For several well-documented reasons, it has been challenging
to develop artificial small molecule inhibitors of protein/protein
complexes. Such reagents are of particular interest for transcription
factor complexes given links between their misregulation and disease.
Here we report parallel approaches to identify regulators of a hypoxia
signaling transcription factor complex, involving the ARNT subunit
of the HIF (Hypoxia Inducible Factor) activator and the TACC3 (Transforming
Acidic Coiled Coil Containing Protein 3) coactivator. In one route,
we used <i>in vitro</i> NMR and biochemical screening to
identify small molecules that selectively bind within the ARNT PAS
(Per-ARNT-Sim) domain that recruits TACC3, identifying KG-548 as an
ARNT/TACC3 disruptor. A parallel, cell-based screening approach previously
implicated the small molecule KHS101 as an inhibitor of TACC3 signaling.
Here, we show that KHS101 works indirectly on HIF complex formation
by destabilizing both TACC3 and the HIF component HIF-1α. Overall,
our data identify small molecule regulators for this important complex
and highlight the utility of pursuing parallel strategies to develop
protein/protein inhibitors