12 research outputs found
Biochemical and structural characterization of C-terminal constructs of bovine soluble guanylate cyclase
C-Terminal Tail Residue Arg1400 Enables NADPH to Regulate Electron Transfer in Neuronal Nitric-Oxide Synthase
The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg1400 in the C-terminal tail regulatory element and the 2′-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg1400 in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent Km for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg1400-2′-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS
Giant viruses of the Megavirinae subfamily possess biosynthetic pathways to produce rare bacterial-like sugars in a clade-specific manner
International audienceThe recent discovery that giant viruses encode proteins related to sugar synthesis and processing paved the way for the study of their glycosylation machinery. We focused on the proposed Megavirinae subfamily, for which glycan-related genes were proposed to code for proteins involved in glycosylation of the layer of fibrils surrounding their icosahedral capsids. We compared sugar compositions and corresponding biosynthetic pathways among clade members using a combination of chemical and bioinformatics approaches. We first demonstrated that Megavirinae glycosylation differs in many aspects from what was previously reported for viruses, as they have complex glycosylation gene clusters made of six and up to 33 genes to synthetize their fibril glycans (biosynthetic pathways for nucleotide-sugars and glycosyltransferases). Second, they synthesize rare amino-sugars, usually restricted to bacteria and absent from their eukaryotic host. Finally, we showed that Megavirinae glycosylation is clade-specific and that Moumouvirus australiensis, a B-clade outsider, shares key features with Cotonvirus japonicus (clade E) and Tupanviruses (clade D). The existence of a glycosylation toolbox in this family could represent an advantageous strategy to survive in an environment where members of the same family are competing for the same amoeba host. This study expands the field of viral glycobiology and raises questions on how Megavirinae evolved such versatile glycosylation machinery
Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors.
CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators
Interfacial Residues Promote an Optimal Alignment of the Catalytic Center in Human Soluble Guanylate Cyclase: Heterodimerization Is Required but Not Sufficient for Activity
Soluble guanylate cyclase (sGC) plays
a central role in the cardiovascular
system and is a drug target for the treatment of pulmonary hypertension.
While the three-dimensional structure of sGC is unknown, studies suggest
that binding of the regulatory domain to the catalytic domain maintains
sGC in an autoinhibited basal state. The activation signal, binding
of NO to heme, is thought to be transmitted via the regulatory and
dimerization domains to the cyclase domain and unleashes the full
catalytic potential of sGC. Consequently, isolated catalytic domains
should show catalytic turnover comparable to that of activated sGC.
Using X-ray crystallography, activity measurements, and native mass
spectrometry, we show unambiguously that human isolated catalytic
domains are much less active than basal sGC, while still forming heterodimers.
We identified key structural elements regulating the dimer interface
and propose a novel role for residues located in an interfacial flap
and a hydrogen bond network as key modulators of the orientation of
the catalytic subunits. We demonstrate that even in the absence of
the regulatory domain, additional sGC domains are required to guide
the appropriate conformation of the catalytic subunits associated
with high activity. Our data support a novel regulatory mechanism
whereby sGC activity is tuned by distinct domain interactions that
either promote or inhibit catalytic activity. These results further
our understanding of heterodimerization and activation of sGC and
open additional drug discovery routes for targeting the NO–sGC–cGMP
pathway via the design of small molecules that promote a productive
conformation of the catalytic subunits or disrupt inhibitory domain
interactions
Interfacial Residues Promote an Optimal Alignment of the Catalytic Center in Human Soluble Guanylate Cyclase: Heterodimerization Is Required but Not Sufficient for Activity
Soluble guanylate cyclase (sGC) plays
a central role in the cardiovascular
system and is a drug target for the treatment of pulmonary hypertension.
While the three-dimensional structure of sGC is unknown, studies suggest
that binding of the regulatory domain to the catalytic domain maintains
sGC in an autoinhibited basal state. The activation signal, binding
of NO to heme, is thought to be transmitted via the regulatory and
dimerization domains to the cyclase domain and unleashes the full
catalytic potential of sGC. Consequently, isolated catalytic domains
should show catalytic turnover comparable to that of activated sGC.
Using X-ray crystallography, activity measurements, and native mass
spectrometry, we show unambiguously that human isolated catalytic
domains are much less active than basal sGC, while still forming heterodimers.
We identified key structural elements regulating the dimer interface
and propose a novel role for residues located in an interfacial flap
and a hydrogen bond network as key modulators of the orientation of
the catalytic subunits. We demonstrate that even in the absence of
the regulatory domain, additional sGC domains are required to guide
the appropriate conformation of the catalytic subunits associated
with high activity. Our data support a novel regulatory mechanism
whereby sGC activity is tuned by distinct domain interactions that
either promote or inhibit catalytic activity. These results further
our understanding of heterodimerization and activation of sGC and
open additional drug discovery routes for targeting the NO–sGC–cGMP
pathway via the design of small molecules that promote a productive
conformation of the catalytic subunits or disrupt inhibitory domain
interactions