Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Quantifying Target Occupancy of Small Molecules Within Living Cells.

The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.

GroEL Ring Separation and Exchange in the Chaperonin Reaction.

The bacterial chaperonin GroEL and its cofactor, GroES, form a nano-cage for a single molecule of substrate protein (SP) to fold in isolation. GroEL and GroES undergo an ATP-regulated interaction cycle to close and open the folding cage. GroEL consists of two heptameric rings stacked back to back. Here, we show that GroEL undergoes transient ring separation, resulting in ring exchange between complexes. Ring separation occurs upon ATP-binding to the trans ring of the asymmetric GroEL:7ADP:GroES complex in the presence or absence of SP and is a consequence of inter-ring negative allostery. We find that a GroEL mutant unable to perform ring separation is folding active but populates symmetric GroEL:GroES2 complexes, where both GroEL rings function simultaneously rather than sequentially. As a consequence, SP binding and release from the folding chamber is inefficient, and E. coli growth is impaired. We suggest that transient ring separation is an integral part of the chaperonin mechanism.

A J-Protein Co-chaperone Recruits BiP to Monomerize IRE1 and Repress the Unfolded Protein Response.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER-protein-folding capacity to restore protein-folding homeostasis. Unfolded proteins activate UPR signaling across the ER membrane to the nucleus by promoting oligomerization of IRE1, a conserved transmembrane ER stress receptor. However, the coupling of ER stress to IRE1 oligomerization and activation has remained obscure. Here, we report that the ER luminal co-chaperone ERdj4/DNAJB9 is a selective IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). In vitro, ERdj4 is required for complex formation between BiP and IRE1LD. ERdj4 associates with IRE1LD and recruits BiP through the stimulation of ATP hydrolysis, forcibly disrupting IRE1 dimers. Unfolded proteins compete for BiP and restore IRE1LD to its default, dimeric, and active state. These observations establish BiP and its J domain co-chaperones as key regulators of the UPR.

Next generation single-molecule techniques: Imaging, labeling, and manipulation in vitro and in cellulo.

Owing to their unique abilities to manipulate, label, and image individual molecules in vitro and in cellulo, single-molecule techniques provide previously unattainable access to elementary biological processes. In imaging, single-molecule fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement in vitro can report on conformational changes and molecular interactions, single-molecule pull-down (SiMPull) can capture and analyze the composition and function of native protein complexes, and single-molecule tracking (SMT) in live cells reveals cellular structures and dynamics. In labeling, the abilities to specifically label genomic loci, mRNA, and nascent polypeptides in cells have uncovered chromosome organization and dynamics, transcription and translation dynamics, and gene expression regulation. In manipulation, optical tweezers, integration of single-molecule fluorescence with force measurements, and single-molecule force probes in live cells have transformed our mechanistic understanding of diverse biological processes, ranging from protein folding, nucleic acids-protein interactions to cell surface receptor function.

A universal allosteric mechanism for G protein activation.

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gßγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gßγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation.

Phylogenic Determinants of Cardiovascular Frailty, Focus on Hemodynamics and Arterial Smooth Muscle Cells.

The evolution of the circulatory system from invertebrates to mammals has involved the passage from an open system to a closed in-parallel system via a closed in-series system, accompanying the increasing complexity and efficiency of life's biological functions. The archaic heart enables pulsatile motion waves of hemolymph in invertebrates, and the in-series circulation in fish occurs with only an endothelium, whereas mural smooth muscle cells appear later. The present review focuses on evolution of the circulatory system. In particular, we address how and why this evolution took place from a closed, flowing, longitudinal conductance at low pressure to a flowing, highly pressurized and bifurcating arterial compartment. However, although arterial pressure was the latest acquired hemodynamic variable, the general teleonomy of the evolution of species is the differentiation of individual organ function, supported by specific fueling allowing and favoring partial metabolic autonomy. This was achieved via the establishment of an active contractile tone in resistance arteries, which permitted the regulation of blood supply to specific organ activities via its localized function-dependent inhibition (active vasodilation). The global resistance to viscous blood flow is the peripheral increase in frictional forces caused by the tonic change in arterial and arteriolar radius, which backscatter as systemic arterial blood pressure. Consequently, the arterial pressure gradient from circulating blood to the adventitial interstitium generates the unidirectional outward radial advective conductance of plasma solutes across the wall of conductance arteries. This hemodynamic evolution was accompanied by important changes in arterial wall structure, supported by smooth muscle cell functional plasticity, including contractility, matrix synthesis and proliferation, endocytosis and phagocytosis, etc. These adaptive phenotypic shifts are due to epigenetic regulation, mainly related to mechanotransduction. These paradigms actively participate in cardio-arterial pathologies such as atheroma, valve disease, heart failure, aneurysms, hypertension, and physiological aging.

Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics.

Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.

In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent in vitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, in situ Foerster resonance energy transfer-fluorescence lifetime imaging (FRET-FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs). In vitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with ß-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.

Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP.

Arg-73 of the RNA endonuclease MazF in Salmonella enterica subsp. arizonae contributes to guanine and uracil recognition in the cleavage sequence.

The sequence-specific endoribonuclease MazF is widely conserved among prokaryotes. Approximately 20 different MazF cleavage sequences have been discovered, varying from three to seven nucleotides in length. Although MazFs from various prokaryotes were found, the cleavage sequences of most MazFs are unknown. Here, we characterized the conserved MazF of Salmonella enterica subsp. arizonae (MazF-SEA). Using massive parallel sequencing and fluorometric assays, we revealed that MazF-SEA preferentially cleaves the sequences U∧ACG and U∧ACU (∧ represents cleavage sites). In addition, we predicted the 3D structure of MazF-SEA using AlphaFold2 and aligned it with the crystal structure of RNA-bound Bacillus subtilis MazF to evaluate RNA interactions. We found Arg-73 of MazF-SEA interacts with RNAs containing G and U at the third position from the cleavage sites (U∧ACG and U∧ACU). We then obtained the mutated MazF-SEA R73L protein to evaluate the significance of Arg-73 interaction with RNAs containing G and U at this position. We also used fluorometric and kinetic assays and showed the enzymatic activity of MazF-SEA R73L for the sequence UACG and UACU was significantly decreased. These results suggest Arg-73 is essential for recognizing G and U at the third position from the cleavage sites. This is the first study to our knowledge to identify a single residue responsible for RNA recognition by MazF. Owing to its high specificity and ribosome-independence, MazF is useful for RNA cleavage in vitro. These results will likely contribute to increasing the diversity of MazF specificity and to furthering the application of MazF in RNA engineering.

Structure of the antenna complex expressed during far-red light photoacclimation in Synechococcus sp. PCC 7335.

Far-red light photoacclimation, or FaRLiP, is a facultative response exhibited by some cyanobacteria that allows them to absorb and utilize lower energy light (700-800 nm) than the wavelengths typically used for oxygenic photosynthesis (400-700 nm). During this process, three essential components of the photosynthetic apparatus are altered: photosystem I, photosystem II, and the phycobilisome. In all three cases, at least some of the chromophores found in these pigment-protein complexes are replaced by chromophores that have red-shifted absorbance relative to the analogous complexes produced in visible light. Recent structural and spectroscopic studies have elucidated important features of the two photosystems when altered to absorb and utilize far-red light, but much less is understood about the modified phycobiliproteins made during FaRLiP. We used single-particle, cryo-EM to determine the molecular structure of a phycobiliprotein core complex comprising allophycocyanin variants that absorb far-red light during FaRLiP in the marine cyanobacterium Synechococcus sp. PCC 7335. The structure reveals the arrangement of the numerous red-shifted allophycocyanin variants and the probable locations of the chromophores that serve as the terminal emitters in this complex. It also suggests how energy is transferred to the photosystem II complexes produced during FaRLiP. The structure additionally allows comparisons with other previously studied allophycocyanins to gain insights into how phycocyanobilin chromophores can be tuned to absorb far-red light. These studies provide new insights into how far-red light is harvested and utilized during FaRLiP, a widespread cyanobacterial photoacclimation mechanism.

Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5ß/B6 and ABCB5ß/B9.

ABCB5 is a member of the ABC transporter superfamily composed of 48 transporters, which have been extensively studied for their role in cancer multidrug resistance and, more recently, in tumorigenesis. ABCB5 has been identified as a marker of skin progenitor cells, melanoma, and limbal stem cells. It has also been associated with multidrug resistance in several cancers. The unique feature of ABCB5 is that it exists as both a full transporter (ABCB5FL) and a half transporter (ABCB5ß). Several studies have shown that the ABCB5ß homodimer does not confer multidrug resistance, in contrast to ABCB5FL. In this study, using three complementary techniques, (1) nanoluciferase-based bioluminescence resonance energy transfer, (2) coimmunoprecipitation, and (3) proximity ligation assay, we identified two novel heterodimers in melanoma: ABCB5ß/B6 and ABCB5ß/B9. Both heterodimers could be expressed in High-Five insect cells and ATPase assays revealed that both functional nucleotide-binding domains of homodimers and heterodimers are required for their basal ATPase activity. These results are an important step toward elucidating the functional role of ABCB5ß in melanocytes and melanoma.

A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination.

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics. The assay also offers a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

Intermediate steps in the formation of neuronal SNARE complexes.

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature-dependent with a reduced concentration at 37 °C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25 and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.

Assembly of CpcL-phycobilisomes.

CpcL-phycobilisomes (CpcL-PBSs) are a reduced type of phycobilisome (PBS) found in several cyanobacteria. They lack the traditional PBS terminal energy emitters, but still show the characteristic red-shifted fluorescence at ~670 nm. We established a method of assembling in vitro a rod-membrane linker protein, CpcL, with phycocyanin, generating complexes with the red-shifted spectral features of CpcL-PBSs. The red-shift arises from the interaction of a conserved key glutamine, Q57 of CpcL in Synechocystis sp. PCC 6803, with a single phycocyanobilin chromophore of trimeric phycocyanin at one of the three ß82-sites. This chromophore is the terminal energy acceptor of CpcL-PBSs and donor to the photosystem(s). This mechanism also operates in PBSs from Acaryochloris marina MBIC11017. We then generated multichromic complexes harvesting light over nearly the complete visible range via the replacement of phycocyanobilin chromophores at sites α84 and ß153 of phycocyanins by phycoerythrobilin and/or phycourobilin. The results demonstrate the rational design of biliprotein-based light-harvesting elements by engineering CpcL and phycocyanins, which broadens the light-harvesting range and accordingly improves the light-harvesting capacity and may be potentially applied in solar energy harvesting.

Histone FRET reports the spatial heterogeneity in nanoscale chromatin architecture that is imparted by the epigenetic landscape at the level of single foci in an intact cell nucleus.

Genome sequencing has identified hundreds of histone post-translational modifications (PTMs) that define an open or compact chromatin nanostructure at the level of nucleosome proximity, and therefore serve as activators or repressors of gene expression. Direct observation of this epigenetic mode of transcriptional regulation in an intact single nucleus, is however, a complex task. This is because despite the development of fluorescent probes that enable observation of specific histone PTMs and chromatin density, the changes in nucleosome proximity regulating gene expression occur on a spatial scale well below the diffraction limit of optical microscopy. In recent work, to address this research gap, we demonstrated that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones core to the nucleosome, is a readout of chromatin nanostructure that can be multiplexed with immunofluorescence (IF) against specific histone PTMs. Here from application of this methodology to gold standard gene activators (H3K4Me3 and H3K9Ac) versus repressors (e.g., H3K9Me3 and H3K27Me), we find that while on average these histone marks do impart an open versus compact chromatin nanostructure, at the level of single chromatin foci, there is significant spatial heterogeneity. Collectively this study illustrates the importance of studying the epigenetic landscape as a function of space within intact nuclear architecture and opens the door for the study of chromatin foci sub-populations defined by combinations of histone marks, as is seen in the context of bivalent chromatin.

Structure and ion-dependent folding of k-junctions.

k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally identified in the structures of Arabidopsis and Escherichia coli thiamine pyrophosphate (TPP) riboswitches, and another called DUF-3268 was tentatively identified from sequence information. In this work we show that the Arabidopsis and E. coli riboswitch k-junctions fold in response to the addition of magnesium or sodium ions, and that atomic mutations that should disrupt key hydrogen bonding interactions greatly impair folding. Using X-ray crystallography, we have determined the structure of the DUF-3268 RNA and thus confirmed that it is a k-junction. It also folds upon the addition of metal ions, though requiring a 40-fold lower concentration of either divalent or monovalent ions. The key difference between the DUF-3268 and riboswitch k-junctions is the lack of nucleotides inserted between G1b and A2b in the former. We show that this insertion is primarily responsible for the difference in folding properties. Finally, we show that the DUF-3268 can functionally substitute for the k-junction in the E. coli TPP riboswitch such that the chimera can bind the TPP ligand, although less avidly.

Oxygenic Photosynthesis in Far-Red Light: Strategies and Mechanisms.

Oxygenic photosynthesis, the process that converts light energy into chemical energy, is traditionally associated with the absorption of visible light by chlorophyll molecules. However, recent studies have revealed a growing number of organisms capable of using far-red light (700-800 nm) to drive oxygenic photosynthesis. This phenomenon challenges the conventional understanding of the limits of this process. In this review, we briefly introduce the organisms that exhibit far-red photosynthesis and explore the different strategies they employ to harvest far-red light. We discuss the modifications of photosynthetic complexes and their impact on the delivery of excitation energy to photochemical centers and on overall photochemical efficiency. Finally, we examine the solutions employed to drive electron transport and water oxidation using relatively low-energy photons. The findings discussed here not only expand our knowledge of the remarkable adaptation capacities of photosynthetic organisms but also offer insights into the potential for enhancing light capture in crops.