Biology subjects

Royer, C. A.

Publications and source records attributed to Royer, C. A..

8 recordsLinked to original sources

bioRxiv2025

Mapping Bidirectional Allosteric Communication in Arf GTPases

The Arf GTPases are eukaryotic signaling proteins implicated in trafficking, motility and membrane remodeling. They must undergo a massive conformational transition in the switch between their inactive, GDP-bound form and their GTP-bound form, competent for downstream signaling. The mechanism of their GDP-to-GTP nucleotide switch implicates a functional molten globule (MG) ensemble. Access to this ensemble and apparent spontaneous switching is modulated by residues in the C-terminal half of the different homologs through a back-to-front allosteric pathway. Here, using high pressure (HP) NMR we show that a mutation in the N-terminal switch region in the front of Arf1 known to modulate spontaneous switching perturbs the stability of residues on a path that stretches from the front to the back. This establishes the existence of a bidirectional and continuous allosteric pathway that passes through the GDP ligand. HP switching studies also demonstrate that the allosteric mechanism controls access to the functional MG state, rather than directly affecting the switching rates. Secondary AbstractThe mechanism of the Arf GTPase nucleotide switch implicates a functional molten globule (MG) ensemble. Switching by the front side of the Arf proteins is controlled by sequences on the back side. Here, using high pressure (HP) NMR we map a bidirectional and continuous allosteric pathway that passes between front and back through the GDP ligand. We also demonstrate that the allosteric mechanism controls access to the functional MG state, rather than directly affecting the switching rates.

biophysics↗

bioRxiv2025

Putting a lid on it: The N-terminal helix of Arf1 inhibits switching via stabilization of the overall native state

The Arf (and Arf-like) GTPases, unlike all other Ras family GTPase members, exhibit a repressed conformation in the inactive, GDP-bound form. The N-terminal helix of Arf GTPases, which is missing in the other Ras family members, caps the switch region, confining it to this repressed state. Nucleotide exchange and activation involve a massive conformational change made possible by the dissociation of the N-terminal helix from the core of the protein. Spontaneous switching in Arfs is enhanced upon deletion of this helix. While the structural and functional role of the N-terminal helix in Arf proteins is well-known, the energetic basis for its effects have not been established. Here we mapped the local stability of the Arf1{Delta}17 variant, deleted for the N-terminal helix, using high pressure biophysical approaches and compared it to that of full-length Arf1. Deletion of the N-terminal helix decreased Arf1 stability across the entire structure. Thus, rather than imposing a specific structural pathway for repression, the N-terminal helix exercises global control of Arf1 stability to repress switching. Statement of SignificanceEnergetic mapping of the N-terminal deletion mutant of Arf1 using high pressure biophysical approaches reveals that this helix maintains the protein in the repressed state unique to Arf and Arf-like GTPases via overall stabilization of the native state rather than unique allosteric communication with the switch region.

biophysics↗

bioRxiv2023

Swi4-dependent SWI4 transcription couples cell size to cell cycle commitment

Growth-dependent accumulation of the limiting SBF transcription factor, composed of Swi4 and Swi6, occurs in G1 phase in budding yeast and is limiting for commitment to division, termed Start. Here we measure size-dependence of Swi4 protein copy number under different genetic contexts using the scanning number and brightness technique. Mutation of SBF binding sites in the SWI4 promoter or disruption of SBF activation resulted in [~]33-50% decrease in Swi4 accumulation rate and concordantly increased cell size at Start. Ectopic inducible expression of Swi4 in G1 phase cells increased production of Swi4 from the endogenous promoter, upregulated transcription of the G1/S regulon, and accelerated Start. Despite the potential for Swi4 positive feedback, G1 phase Swi4 accumulation was linear unless the Whi5 transcriptional repressor was inactivated. A threshold model in which Swi4 titrates SBF binding sites in G1/S promoters predicted the effects of nutrients, ploidy, and G1/S regulatory mutations on cell size. These results exemplify how transcription factor auto-production can contribute to a cell state transition.

cell biology↗

bioRxiv2021

Structural Evolution of the Ancient Enzyme, Dissimilatory Sulfite Reductase

Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme accompanied diversification of sulfate/sulfite reducing organisms (SRO) into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving SRO lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.

biophysics↗

bioRxiv2020

Hepatitis C Virus Drugs Simeprevir and Grazoprevir Synergize with Remdesivir to Suppress SARS-CoV-2 Replication in Cell Culture

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2 virus. Here we assess ten available HCV protease inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS- CoV-2 Mpro and HCV NS3/4A proteases, and virtual docking experiments show that all ten HCV drugs can potentially bind into the Mpro binding cleft. Seven of these HCV drugs inhibit SARS-CoV-2 Mpro protease activity, while four dock well into the PLpro substrate binding cleft and inhibit PLpro protease activity. These same seven HCV drugs inhibit SARS-CoV-2 virus replication in Vero and/or human cells, demonstrating that HCV drugs that inhibit Mpro, or both Mpro and PLpro, suppress virus replication. Two HCV drugs, simeprevir and grazoprevir synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, thereby increasing remdesivir inhibitory activity as much as 10-fold. HighlightsO_LISeveral HCV protease inhibitors are predicted to inhibit SARS-CoV-2 Mpro and PLpro. C_LIO_LISeven HCV drugs inhibit Mpro enzyme activity, four HCV drugs inhibit PLpro. C_LIO_LISeven HCV drugs inhibit SARS-CoV-2 replication in Vero and/or human cells. C_LIO_LIHCV drugs simeprevir and grazoprevir synergize with remdesivir to inhibit SARS- CoV-2. C_LI eTOC blurbBafna, White and colleagues report that several available hepatitis C virus drugs inhibit the SARS-CoV-2 Mpro and/or PLpro proteases and SARS-CoV-2 replication in cell culture. Two drugs, simeprevir and grazoprevir, synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir antiviral activity as much as 10-fold. O_FIG O_LINKSMALLFIG WIDTH=185 HEIGHT=200 SRC="FIGDIR/small/422511v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1c12181org.highwire.dtl.DTLVardef@7ed993org.highwire.dtl.DTLVardef@1fe56aaorg.highwire.dtl.DTLVardef@ebc34e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Nsr1, a nitrogen source-regulated microprotein, confers an alternative mechanism of G1/S transcriptional activation in budding yeast

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon mTORCl inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogeny conditions. Author SummarUnicellular microorganisms must adapt to ever-changing nutrient conditions and hence must adjust cell growth and proliferation to maximize fitness. In the budding yeast Saccharomyces cerevisiae, commitment to cell division, termed Start, is heavily influenced by nutrient availability. Our understanding of how Start is activated is based mainly on experiments carried out under rich nutrient conditions. To identify potential new Start regulators specific to poor nutrient environments, we screened for genes able to bypass a genetic Start arrest caused by loss of the G1 cyclin Cln3 and the transcriptional activator Bck2. This screen uncovered YLR053c, which we renamed NRS1 for Nitrogen-Responsive Start regulator. Sequence analysis across yeast species indicated that Nrs1 is a recently-evolved microprotein. We showed that NRS1 is nutrient- and cell cycle-regulated, and directly binds the main G1/S transcription factor complex SBF. We demonstrated that Nrs1 has an intrinsic trans-activation activity and provided genetic evidence to suggest that Nrs1 can bypass the requirement for normal Cln3-dependent activation of G1/S transcription. These results uncover a new mechanism of Start activation and illustrate how microproteins can rapidly emerge to rewire fundamental cellular processes.

cell biology↗

bioRxiv2019

Quantitative high-resolution imaging of live microbial cells at high hydrostatic pressure

The majority of the Earths microbial biomass exists in the Deep Biosphere, in the deep ocean and within the Earths crust. While other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high-pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold pressure on the Earths surface. The mechanisms of pressure adaptation are also important to in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here we describe implementation of a high-pressure sample chamber with a 2-photon scanning microscope system allowing for the first time, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for Fluorescence Lifetime Imaging Microscopy with Phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon, Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning Number and Brightness (sN&B) and Raster scanning Imaging Correlation Spectroscopy (RICS)) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in E. coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. SIGNIFICANCEThe majority of the Earths microbial biomass exists in high-pressure environments where pressures can reach over 100 MPa. The molecular mechanisms that allow microbes to flourish under such extreme conditions remain to be discovered. The high pressure, high resolution imaging system presented here revealed pressure dependent changes in metabolism and protein interactions in live microbial cells, demonstrating great promise for understanding deep life.

microbiology↗

bioRxiv2019

G1/S transcription factors assemble in discrete clusters that increase in number as cells grow

The spatio-temporal organization of transcription factor (TF)-promoter interactions is critical for the coordination of transcriptional programs. In budding yeast, the main G1/S transcription factors, SBF and MBF, are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the question of how SBF/MBF are dynamically distributed across the G1/S regulon. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits revealed that 85% were organized into discrete clusters containing [~]8 copies regardless of cell size, while the number of clusters increased with growth. Stochastic simulations with a mathematical model based on co-localization of promoters in clusters recapitulated observed cluster behavior. A prediction of the model that SBF/MBF should exhibit both fast and slow dynamics was confirmed in PALM experiments on live cells. This spatio-temporal organization of the TFs that activate the G1/S regulon may help coordinate commitment to division.

cell biology↗