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bioRxiv2020

The Nucleosome Remodeling and Deacetylase complex has an asymmetric, dynamic, and modular architecture

The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for development in complex animals but has been refractory to biochemical analysis. We present the first integrated analysis of the architecture of the native mammalian NuRD complex, combining quantitative mass spectrometry, covalent cross-linking, protein biochemistry and electron microscopy. NuRD is built around a 2:2:4 pseudo-symmetric deacetylase module comprising MTA, HDAC and RBBP subunits. This module interacts asymmetrically with a remodeling module comprising one copy each of MBD, GATAD2 and CHD subunits. The previously enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the ATP-dependent CHD remodeler. Unexpectedly, the MTA-MBD interaction acts as a point of functional switching. The transcriptional regulator PWWP2A modulates NuRD assembly by competing directly with MBD for binding to the MTA-HDAC-RBBP subcomplex, forming a moonlighting PWWP2A-MTA-HDAC-RBBP complex that likely directs deacetylase activity to PWWP2A target sites. Taken together, our data describe the overall architecture of the intact NuRD complex and reveal aspects of its structural dynamics and functional plasticity.

biochemistry↗

bioRxiv2020

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a fingerprint of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (g-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

biochemistry↗

bioRxiv2020

Water as a reactant in the differential expression of proteins in cancer

How the abundances of proteins are shaped by tumor microenvironments, such as hypoxic conditions and higher water content compared to normal tissues, is an important question for cancer biochemistry. Compositional analysis of more than 250 datasets for differentially expressed proteins compiled from the literature reveals a higher stoichiometric hydration state in multiple cancer types compared to normal tissue; this trend is also evident in pan-cancer transcriptomic and proteomic datasets from The Cancer Genome Atlas and Human Protein Atlas. These findings support the notion of a basic physicochemical link between increased water content in tumors and the patterns of gene and protein expression in cancer. The generally increased hydration state is juxtaposed with a wide spectrum of carbon oxidation states of differentially expressed proteins, which may be associated with different gene ages, host tissue properties and metabolic features of specific cancer types.

biochemistry↗

bioRxiv2020

The interplay between Bag-1, Hsp70, and Hsp90 reveals that inhibiting Hsp70 rebinding is essential for Glucocorticoid Receptor activity

The glucocorticoid receptor (GR), like many signaling proteins requires Hsp90 for sustained activity. Previous biochemical studies revealed that the requirement for Hsp90 is explained by its ability to reverse Hsp70-mediated inactivation of GR through a complex process requiring both cochaperones and Hsp90 ATP hydrolysis. How ATP hydrolysis on Hsp90 enables GR reactivation is unknown. The canonical mechanism of client release from Hsp70 requires ADP:ATP exchange, which is normally rate limiting. Here we show that independent of ATP hydrolysis, Hsp90 acts as an Hsp70 nucleotide exchange factor (NEF) to accelerate ADP dissociation, likely coordinating GR transfer from Hsp70 to Hsp90. As Bag-1 is a canonical Hsp70 NEF that can also reactivate Hsp70:GR, the impact of these two NEFs was compared. Simple acceleration of Hsp70:GR release was insufficient for GR reactivation as Hsp70 rapidly re-binds and re-inactivates GR. Instead, inhibition of GR re-inactivation by Hsp70 is critical. This can be accomplished by high non-physiological Bag-1 concentrations, which also inhibit Hsp70:ATP binding. In contrast, in an ATP-hydrolysis dependent process, Hsp90 plays a unique role by kinetically partitioning GR into a state that can bind ligand, but is protected from Hsp70 inactivation, thus allowing GR to be activated by its ligand but still able to re-enter the chaperone cycle. At physiologic concentrations, Bag-1 works synergistically with Hsp90 to accelerate the first rate-limiting step in GR reactivation. The net effect is that the chaperone machinery cyclically dictates the on and off rates for GR ligand, providing a timer controlling the persistence of activated GR. Significance StatementThe glucocorticoid receptor (GR) is an essential transcription regulatory factor. Like many signaling proteins, GR activity is regulated by two essential molecular chaperones: Hsp90 and Hsp70. Functioning like a toggle switch, Hsp70 first inactivates GR, and then Hsp90 reactivates GR in an Hsp90 ATP hydrolysis dependent manner. Here, an intricate set of biochemistry experiments uncover fundamental principles governing how these chaperone systems collaboratively regulate GR activity. While Hsp90 promotes GR release from Hsp70 by modulating Hsp70s nucleotide state, this occurs independently of Hsp90 ATP hydrolysis. Instead, ATP hydrolysis on Hsp90 facilitates a second essential reactivation step resulting in an Hsp90-bound GR state that protects GR from Hsp70 re-inactivation. A kinetic partitioning model best describes chaperone modulation of GRs activity.

biochemistry↗

bioRxiv2020

The SARS-CoV-2 conserved macrodomain is a highly efficient ADP-ribosylhydrolase enzyme

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low M affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. IMPORTANCESARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 900 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

biochemistry↗

bioRxiv2020

Preferred Conformations in the Intrinsically Disordered Region of Human CPEB3 Explain its Role in Memory Consolidation

While implicated in neurodegenerative diseases, amyloids are also essential to some physiological processes, including memory consolidation by neuronal-specific isoforms of the Cytoplasmic Polyadenylation Element Binding (CPEB) protein family. CPEB mediates memory persistence by the formation of self-sustaining amyloid assemblies through its intrinsically disordered region (IDR). Here, we characterize the atomic level conformation and ps-ns dynamics of the 426-residue IDR of human CPEB3 (hCPEB3), which has been associated with episodic memory in humans, by NMR spectroscopy. We found that the first 29 residues: M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical+disordered motif. Residues 86-93: P83QQPPPP93, and 166-175: P166PPPAPAPQP175 form polyproline II (PPII) helices. While the (VG)5 repeat motif is completely disordered, residues 200-250 adopt three partially populated -helices. Residues 345-355, which comprise the nuclear localization signal (NLS), form a modestly populated -helix and border a phosphoTyr which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids. Besides revealing some aspects of the molecular basis of memory, these findings could aid the future development of treatments for post-traumatic stress disorder. Areas: Biophysics, Structural Biology, Biochemistry & Neurosciences.

biochemistry↗

bioRxiv2020

Hydrazines as versatile chemical biology probes and drug-discovery tools for cofactor-dependent enzymes

Known chemoproteomic probes generally use warheads that tag a single type of amino acid or modified form thereof to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electron-poor enzyme cofactors, transient intermediates and chemically-labile regulatory modifications, but probes for such species are underdeveloped. Here, we have innovated a versatile class of chemoproteomic probes for this less charted hemisphere of the proteome by using hydrazine as the common chemical warhead. Its electron-rich nature allows it to react by both polar and radicaloid mechanisms and to target multiple, pharmacologically important functional classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment can be blocked by active-site-directed inhibitors, and elaboration of the warhead supports connection of a target to a lead compound. The capacity of substituted hydrazines to profile, discover and inhibit diverse cofactor-dependent enzymes enables cell and tissue imaging and makes this platform useful for enzyme and drug discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/154864v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1e1a54forg.highwire.dtl.DTLVardef@12be680org.highwire.dtl.DTLVardef@129e027org.highwire.dtl.DTLVardef@3b2317_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Kinetics and Fidelity of Psychrophilic DNA Polymerases

The discovery of extremophiles helped enable the development of groundbreaking technology such as polymerase chain reaction. Temperature variation is often an essential step of these technology platforms, but the effect of temperature on the error rate of polymerases from different origins is under-explored. Here, we applied high-throughput sequencing to profile the error rates of DNA polymerases from psychrophilic, mesophilic, and thermophilic origins with single-molecule resolution. We found that reaction temperature substantially increases substitution and deletion error rates of psychrophilic and mesophilic DNA polymerases. Our motif analysis shows that the substitution error profiles cluster according to phylogenetic similarity of polymerases, not reaction temperature, thus suggesting that reaction temperature increases global error rate of polymerases independent of sequence context. Intriguingly, we also found that the DNA polymerase I of a psychrophilic bacteria exhibits higher polymerization activity than its mesophilic ortholog across all temperature ranges, including down to -19{degrees}C which is well below the freezing temperature of water. Our results provide a useful reference for how reaction temperature, a crucial parameter of biochemistry, can affect DNA polymerase fidelity in organisms adapted to a wide range of thermal environments.

biochemistry↗

bioRxiv2020

D-Serine, an emerging biomarker of kidney diseases, is a hidden substrate of sodium-coupled monocarboxylate transporters

Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While D-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal D-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a D-serine transporter previously uncharacterized in the kidney and discovered D-serine as a noncanonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal D-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.

biochemistry↗

bioRxiv2020

Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases

RNA helicases and ubiquitin E3 ligases mediate many critical functions within cells, but their actions have been studied largely in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryo-electron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity-driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass-spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses thus reveal not only conserved substrates for TRIM proteins but also unexpectedly deep evolutionary connections between TRIM proteins and RNA helicases, thereby linking ubiquitin and RNA biology throughout animal evolution.

biochemistry↗

bioRxiv2020

A Liquid-to-Solid Phase Transition Enhances the Catalytic Activity of SARM1

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is a neuronally expressed NAD+ glycohydrolase whose activity is increased in response to various stressors. The consequent depletion of NAD+ triggers axonal degeneration (i.e., Wallerian degeneration), which is a characteristic feature of neurological diseases, including peripheral neuropathies and traumatic brain injury. Notably, SARM1 knockout mice show minimal degeneration in models of peripheral neuropathy and traumatic brain injury, making SARM1 a promising therapeutic target. However, the development of SARM1 inhibitors has been challenging as the purified enzyme is largely inactive. Herein, we report that SARM1 activity is increased [~]2000-fold by a liquid-to-solid phase transition. These findings provide critical insights into SARM1 biochemistry with important implications for the situation in vivo. Moreover, they will facilitate the discovery of novel SARM1-targeted therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/272377v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@d9c222org.highwire.dtl.DTLVardef@588637org.highwire.dtl.DTLVardef@1d3a3d4org.highwire.dtl.DTLVardef@1a8e058_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Understanding the compositional changes in LB Lennox medium during growth of Escherichia coli DH5α in a 1L bioreactor

Compositional changes in growth medium represents dynamic interplay between cell growth, biomass formation, and energy maintenance, with concomitant decrease in nutrients and increase in secreted metabolites and metabolic byproducts. Such information is important for quantifying microbial physiological response both at the population and cellular level, with respect to understanding subtle differences in microbial growth response, as well as supporting model building efforts in metabolic engineering. With the desire to understand molecular weight changes in components of LB Lennox medium as well as broth fractions present during cultivation of Escherichia coli DH5 (ATCC 53868) at 37 {degrees}C, 400 rpm stirring and 1 VVM aeration in a 1 L bioreactor, this study used a combination of gel filtration chromatography (GFC) and reversed phase high performance liquid chromatography (RP-HPLC) for determining changes to molecular weight of different fractions of the growth medium. Experiment results revealed the difficulty of fractionating the culture broth with RP-HPLC, where no distinct peaks of narrow retention time width were obtained. More importantly, the column used for GFC was unable to differentiate small molecular weight changes on the order of a few tens to few hundred Da through a refractive index detector. Together, GFC and RP-HPLC highlighted the difficulty of fractionating LB Lennox culture broth into different distinct fractions. Finally, the study validated the use of 194 nm as detection wavelength for visualizing the chromatogram of LB Lennox medium eluted from a C-18 reversed phase column during liquid chromatography. Collectively, GFC and RP-HPLC could not fractionate LB Lennox broth of E. coli DH5 into distinct fractions for further analysis by identification techniques such as mass spectrometry. Given the inherent complexity of complex medium such as LB Lennox, clean separation of the medium into every component with high purity may be impossible. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=47 SRC="FIGDIR/small/311373v1_ufig1.gif" ALT="Figure 1"> View larger version (7K): org.highwire.dtl.DTLVardef@2d0861org.highwire.dtl.DTLVardef@814ebcorg.highwire.dtl.DTLVardef@158328borg.highwire.dtl.DTLVardef@15e6844_HPS_FORMAT_FIGEXP M_FIG C_FIG Short descriptionBroad elution profile and lack of distinct peaks in chromatogram of LB Lennox broth at 6 hours post inoculation with Escherichia coli DH5 (ATCC 53868), after attempted fractionation by C-18 reversed phase high performance liquid chromatography (RP-HPLC), revealed that separation could not be achieved with a complex mixture such as a microbiology broth. Specifically, while the objective of understanding compositional changes to culture medium during growth presents tremendous opportunities for determining the dynamic conversion of nutrients into metabolites and byproducts, difficulty of profiling all chemical constituents in a complex broth mixture meant that current model building efforts for metabolic engineering remains primitive with respect to the consortia of metabolic reactions occurring in situ at the cellular level. Subject areasbiotechnology, biochemistry, cell biology, microbiology, analytical chemistry

biochemistry↗

bioRxiv2020

Epistasis on the stability landscape of de novo TIM barrels explored by a modular design approach

The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. High conformational stability lowers protein sensitivity to mutations and changes in the environment; thus, understanding and manipulating protein stability will expand the applications of de novo proteins. Since the ({beta}/)8-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design. Significance StatementDe novo protein design expands our knowledge about protein structure and stability. The TIM barrel is a highly relevant fold used in nature to host a rich variety of catalytic functions. Here, we follow a modular approach to design and characterize a collection of de novo TIM barrels and subjected them to a thorough folding analysis. Non-additive effects modulate the increase in stability when different regions of the barrel are mutated, showing a wide variety of thermodynamic properties that allow them to navigate an unexplored region of the stability landscape found in natural TIM barrels. The design of stable proteins increases the applicability of de novo proteins and provides crucial information on the molecular determinants that modulate structure and stability. One Sentence SummaryA family of designed TIM barrels with diverse thermodynamic properties shows epistatic effects on its stability landscape.

biochemistry↗

bioRxiv2020

Mutations in SKI in Shprintzen-Goldberg syndrome lead to attenuated TGF-β responses through SKI stabilization

Shprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-{beta} signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional corepressor SKI, which is a negative regulator of TGF-{beta} signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-{beta} responses, in both knockin cells expressing an SGS mutation, and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-{beta}-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

biochemistry↗

bioRxiv2020

CaMKII binds both substrates and effectors at the active site

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. Once activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition to well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A longstanding model invokes two distinct S- and T-sites. If an interactor binds at the T-site, it will preclude autoinhibition and allow substrates to be phosphorylated at the S-site. Here, we specifically test this model with X-ray crystallography, molecular dynamics simulations, and biochemistry. Our data are inconsistent with this model. Co-crystal structures of four different activators or substrates show that they all bind to a single continuous site across the kinase domain. We propose a mechanistic model that persistent CaMKII activity is facilitated by high affinity binding partners, which kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation.

biochemistry↗

bioRxiv2020

Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients1-4. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-25-7. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls6,8. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3-exonuclease that recognizes mismatches9,10. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

biochemistry↗

bioRxiv2020

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 GTP binding and the dissociation of G from the G{beta}{gamma} subunits. Structural studies have revealed the molecular basis for subunit association with receptors, RGS proteins and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We used cell signaling assays, MD simulations, biochemistry and structural analysis 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 G{beta}{gamma} dimer. Replacement of network residues prevents subunit dissociation, regardless of agonist or GTP binding. These findings reveal the molecular basis for the final committed step of G protein activation. HIGHLIGHTSO_LIReceptors promote GTP-GDP exchange and dissociation of G protein and {beta}{gamma} subunits C_LIO_LIWe find an allosteric network linking the {gamma} phosphate of GTP with release of G{beta}{gamma} C_LIO_LIThe network consists of a conserved Gly-Arg-Glu "activation triad" C_LIO_LITriad mutations prevent subunit dissociation, regardless of agonist or GTP binding C_LIO_LITriad mutations are responsible for human endocrine and neurological disorders C_LI

biochemistry↗

bioRxiv2020

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK, a microfluidic platform for high-throughput expression, purification, and characterization of >1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA, we performed >670,000 reactions to determine >5000 kinetic and physical constants for multiple substrates and inhibitors. These constants allowed us to uncover extensive kinetic partitioning to a misfolded state and isolate catalytic effects, revealing spatially contiguous "regions" of residues linked to particular aspects of function. These regions included active-site proximal residues but also extended to the enzyme surface, providing a map of underlying architecture that could not be derived from existing approaches. HT-MEK, using direct and coupled fluorescent assays, has future applications to a wide variety of problems ranging from understanding molecular mechanisms to medicine to engineering and design. One Sentence SummaryHT-MEK, a microfluidic platform for high-throughput, quantitative biochemistry, reveals enzyme architectures shaping function.

biochemistry↗