CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage.

Type III-E CRISPR-Cas effectors, of which Cas7-11 is the first, are single proteins that cleave target RNAs without nonspecific collateral cleavage, opening new possibilities for RNA editing. Biochemical experiments combined with amide hydrogen-deuterium exchange (HDX-MS) experiments provide a first glimpse of the conformational dynamics of apo Cas7-11. HDX-MS revealed the backbone comprised of the four Cas7 zinc-binding RRM folds are well-folded but insertion sequences are highly dynamic and fold upon binding crRNA. The crRNA causes folding of disordered catalytic loops and ß-hairpins, stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. Target RNA binding causes only minor ordering around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot fully process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces target RNA cleavage. The insertion domain shows the most ordering upon binding of mature crRNA. Finally, we show a crRNA-induced conformational change in one of the TPR-CHAT binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding. The results provide the first glimpse of the apo state of Cas7-11 and reveal how its structure and function are regulated by crRNA binding.

Diversity of structure and function in Cullin E3 ligases.

The cellular process by which the protein ubiquitin (Ub) is covalently attached to a protein substrate involves Ub activating (E1s) and conjugating enzymes (E2s) that work together with a large variety of E3 ligases that impart substrate specificity. The largest family of E3s is the Cullin-RING ligase (CRL) family which utilizes a wide variety of substrate receptors, adapter proteins, and cooperating ligases. Cryo-electron microscopy (cryoEM) has revealed a wide variety of structures which suggest how Ub transfer occurs. Hydrogen deuterium exchange mass spectrometry (HDXMS) has revealed the role of dynamics and expanded our knowledge of how covalent NEDD8 modification (neddylation) activates the CRLs, particularly by facilitating cooperation with additional RING-between-RING ligases to transfer Ub.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant unusually preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employ static and dynamic structural methods and observe that, compared to R132H, the R132Q active site adopts a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling reveals a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

The Light Chain Allosterically Enhances the Protease Activity of Murine Urokinase-Type Plasminogen Activator.

The active form of the murine urokinase-type plasminogen activator (muPA) is formed by a 27-residue disordered light chain connecting the amino-terminal fragment (ATF) with the serine protease domain. The two chains are tethered by a disulfide bond between C1CT in the disordered light chain and C122CT in the protease domain. Previous work showed that the presence of the disordered light chain affected the inhibition of the protease domain by antibodies. Here we show that the disordered light chain induced a 3.7-fold increase in kcat of the protease domain of muPA. In addition, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and accelerated molecular dynamics (AMD) were performed to identify the interactions between the disordered light chain and the protease domain. HDX-MS revealed that the light chain is contacting the 110s, the turn between the ß10- and ß11-strand, and the ß7-strand. A reduction in deuterium uptake was also observed in the activation loop, the 140s loop and the 220s loop, which forms the S1-specificty pocket where the substrate binds. These loops are further away from where the light chain seems to be interacting with the protease domain. Our results suggest that the light chain most likely increases the activity of muPA by allosterically favoring conformations in which the specificity pocket is formed. We propose a model by which the allostery would be transmitted through the ß-strands of the ß-barrels to the loops on the other side of the protease domain.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

The multifunctional role of intrinsic disorder in NF-κB signaling.

The nuclear factor-κB (NF-κB) transcription activation system involves disordered regions of both the NF-κB dimers and their inhibitors, the IκBs. The system is well-studied both at the cellular and biophysical levels affording a unique opportunity to compare and contrast the conclusions from both types of experiments. Through a combination of both experiments and theory, we have discovered that the RelA/p50 heterodimer and its inhibitor IκBα operate under kinetic control. Intrinsically disordered parts of both proteins are directly involved in temporal control and their folding and unfolding determines the rates of various processes. In this review, we show how the dynamic state of the intrinsically disordered sequences define the rates of intracellular processes.

The autoactivation of human single-chain urokinase-type plasminogen activator (uPA).

Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used hydrogen deuterium exchange mass spectrometry to characterize the dynamic differences between the inactive single-chain uPA (scuPA) and its active form two-chain uPA (tcuPA). The results show that the C-terminal ß-barrel and the area around the new N terminus have significantly reduced dynamics in tcuPA as compared with scuPA. We also show that the zymogen scuPA is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the tcuPA can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation.

Dynamic allostery in thrombin-a review.

Thrombin is a serine protease that catalyzes a large number of different reactions including proteolytic cleave of fibrinogen to make the fibrin clot (procoagulant activity), of the protease activated receptors (for cell signaling) and of protein C generating activated protein C (anticoagulant activity). Thrombin has an effector binding site called the anion binding exosite 1 that is allosterically coupled to the active site. In this review, we survey results from thermodynamic characterization of the allosteric coupling as well as hydrogen-deuterium exchange mass spectrometry to reveal which parts of the thrombin structure are changed upon effector binding and/or mutagenesis, and finally NMR spectroscopy to characterize the different timescales of motions elicited by the effectors. We also relate the experimental work to computational network analysis of the thrombin-thrombomodulin complex.

Site-specific incorporation of biophysical probes into NF-ĸB with non-canonical amino acids.

The transcription factor NF-ĸB is a central mediator of immune and inflammatory responses. To understand the regulation of NF-ĸB, it is important to probe the underlying thermodynamics, kinetics, and conformational dynamics of the NF-ĸB/IĸBα/DNA interaction network. The development of genetic incorporation of non-canonical amino acids (ncAA) has enabled the installation of biophysical probes into proteins with site specificity. Recent single-molecule FRET (smFRET) studies of NF-ĸB with site-specific labeling via ncAA incorporation revealed the conformational dynamics for kinetic control of DNA-binding mediated by IĸBα. Here we report the design and protocols for incorporating the ncAA p-azidophenylalanine (pAzF) into NF-ĸB and site-specific fluorophore labeling with copper-free click chemistry for smFRET. We also expanded the ncAA toolbox of NF-ĸB to include p-benzoylphenylalanine (pBpa) for UV crosslinking mass spectrometry (XL-MS) and incorporated both pAzF and pBpa into the full-length NF-ĸB RelA subunit which includes the intrinsically disordered transactivation domain.

Capturing the Dynamic Conformational Changes of Human Isocitrate Dehydrogenase 1 (IDH1) upon Ligand and Metal Binding Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Human isocitrate dehydrogenase 1 (IDH1) is a highly conserved metabolic enzyme that catalyzes the interconversion of isocitrate and α-ketoglutarate. Kinetic and structural studies with IDH1 have revealed evidence of striking conformational changes that occur upon binding of its substrates, isocitrate and NADP+, and its catalytic metal cation. Here, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to build a comprehensive map of the dynamic conformational changes experienced by IDH1 upon ligand binding. IDH1 proved well-suited for HDX-MS analysis, allowing us to capture profound changes in solvent accessibility at substrate binding sites and at a known regulatory region, as well as at more distant local subdomains that appear to support closure of this protein into its active conformation. HDX-MS analysis suggested that IDH1 is primarily purified with NADP(H) bound in the absence of its metal cation. Subsequent metal cation binding, even in the absence of isocitrate, was critical for driving large conformational changes. WT IDH1 folded into its fully closed conformation only when the full complement of substrates and metal was present. Finally, we show evidence supporting a previously hypothesized partially open conformation that forms prior to the catalytically active state, and we propose this conformation is driven by isocitrate binding in the absence of metal.

An intrinsically disordered transcription activation domain increases the DNA binding affinity and reduces the specificity of NFκB p50/RelA.

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with coactivators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. Whether these results can be generalized to more TADs is not clear. Here, we biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the TAD's influence on NFκB-DNA interactions. In solution, we show the RelA TAD is disordered but compact, with helical tendency in two regions that interact with coactivators. We determined that the presence of the TAD increased the stoichiometry of NFκB-DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. In addition, we measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it also increased the affinity for nonspecific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. In contrast, previous studies have generally reported that TADs decrease DNA-binding affinity and increase sequence specificity. Our results reveal a novel function of the RelA TAD in promoting binding to nonconsensus DNA, which sheds light on previous observations of extensive nonconsensus DNA binding by NFκB in vivo in response to strong inflammatory signals.

Allosteric couplings upon binding of RfaH to transcription elongation complexes.

In every domain of life, NusG-like proteins bind to the elongating RNA polymerase (RNAP) to support processive RNA synthesis and to couple transcription to ongoing cellular processes. Structures of factor-bound transcription elongation complexes (TECs) reveal similar contacts to RNAP, consistent with a shared mechanism of action. However, NusG homologs differ in their regulatory roles, modes of recruitment, and effects on RNA synthesis. Some of these differences could be due to conformational changes in RNAP and NusG-like proteins, which cannot be captured in static structures. Here, we employed hydrogen-deuterium exchange mass spectrometry to investigate changes in local and non-local structural dynamics of Escherichia coli NusG and its paralog RfaH, which have opposite effects on expression of xenogenes, upon binding to TEC. We found that NusG and RfaH regions that bind RNAP became solvent-protected in factor-bound TECs, whereas RNAP regions that interact with both factors showed opposite deuterium uptake changes when bound to NusG or RfaH. Additional changes far from the factor-binding site were observed only with RfaH. Our results provide insights into differences in structural dynamics exerted by NusG and RfaH during binding to TEC, which may explain their different functional outcomes and allosteric regulation of transcriptional pausing by RfaH.

Target binding triggers hierarchical phosphorylation of human Argonaute-2 to promote target release.

Argonaute (Ago) proteins play a central role in post-transcriptional gene regulation through RNA interference (RNAi). Agos bind small RNAs (sRNAs) including small interfering RNAs (siRNAs) and microRNAs (miRNAs) to form the functional core of the RNA-induced silencing complex (RISC). The sRNA is used as a guide to target mRNAs containing either partially or fully complementary sequences, ultimately leading to downregulation of the corresponding proteins. It was previously shown that the kinase CK1α phosphorylates a cluster of residues in the eukaryotic insertion (EI) of Ago, leading to the alleviation of miRNA-mediated repression through an undetermined mechanism. We show that binding of miRNA-loaded human Ago2 to target RNA with complementarity to the seed and 3' supplementary regions of the miRNA primes the EI for hierarchical phosphorylation by CK1α. The added negative charges electrostatically promote target release, freeing Ago to seek out additional targets once it is dephosphorylated. The high conservation of potential phosphosites in the EI suggests that such a regulatory strategy may be a shared mechanism for regulating miRNA-mediated repression.

Proteins are the chemical 'workhorses' of the cell: some help maintain a cell's shape or structure, while others carry out the chemical reactions necessary for life. Organisms therefore need to keep tight control over the production of proteins in their cells, so that the right amount of each protein is made at the right time, in the right place. Instructions for making new proteins are encoded in a type of molecule called messenger RNA. Each messenger RNA contains the instructions for one protein, which are then 'read' and carried out by special cellular machinery called ribosomes. The cell can control how much protein it produces by regulating both the levels of different messenger RNA and the amount of protein ribosomes are allowed to make from those instructions. The main way to regulate the levels of messenger RNA is through their transcription from the genome. However, this needs fine tuning. Cells can do this in a highly specific way using molecules called microRNAs. A microRNA works by directing a protein called Argonaute to the messenger RNA that it targets. Once Argonaute arrives, it can call in additional 'helper proteins' to shut down, or reduce, protein production from that messenger RNA, or alternatively to break down the messenger RNA altogether. Cells can use an enzyme called CK1α to attach bulky chemical groups onto a specific part of the Argonaute protein, in a reaction termed phosphorylation. The ability to carry out this reaction (and to reverse it) also seems to be important for microRNAs to do their job properly, but why has remained unknown. Bibel et al. wanted to determine what triggers CK1α to phosphorylate Argonaute, and how this affects interactions between microRNAs, Argonaute and their target messenger RNAs. A series of 'test tube' experiments looked at the interaction between purified CK1α and Argonaute under different conditions. These demonstrated that CK1α could only carry out its phosphorylation reaction when Argonaute was already interacting with a microRNA and its corresponding messenger RNA. Further measurements revealed that phosphorylation of Argonaute made it detach from the messenger RNA more quickly. This suggests that phosphorylation might be a way to let Argonaute seek out new messenger RNAs after blocking protein production at its first 'target'. These results shed new light on a fundamental mechanism that cells use to control protein production. Bibel et al. propose that this mechanism may be shared across many different species and could one day help guide the development of new medical therapies based on microRNAs.

Dimer dissociation is a key energetic event in the fold-switch pathway of KaiB.

Cyanobacteria possesses the simplest circadian clock, composed of three proteins that act as a phosphorylation oscillator: KaiA, KaiB, and KaiC. The timing of this oscillator is determined by the fold-switch of KaiB, a structural rearrangement of its C-terminal half that is accompanied by a change in the oligomerization state. During the day, KaiB forms a stable tetramer (gsKaiB), whereas it adopts a monomeric thioredoxin-like fold during the night (fsKaiB). Although the structures and functions of both native states are well studied, little is known about the sequence and structure determinants that control their structural interconversion. Here, we used confinement molecular dynamics (CCR-MD) and folding simulations using structure-based models to show that the dissociation of the gsKaiB dimer is a key energetic event for the fold-switch. Hydrogen-deuterium exchange mass spectrometry (HDXMS) recapitulates the local stability of protein regions reported by CCR-MD, with both approaches consistently indicating that the energy and backbone flexibility changes are solely associated with the region that fold-switches between gsKaiB and fsKaiB and that the localized regions that differentially stabilize gsKaiB also involve regions outside the dimer interface. Moreover, two mutants (R23C and R75C) previously reported to be relevant for altering the rhythmicity of the Kai clock were also studied by HDXMS. Particularly, R75C populates dimeric and monomeric states with a deuterium incorporation profile comparable to the one observed for fsKaiB, emphasizing the importance of the oligomerization state of KaiB for the fold-switch. These findings suggest that the information necessary to control the rhythmicity of the cyanobacterial biological clock is, to a great extent, encoded within the KaiB sequence.

How Thrombomodulin Enables W215A/E217A Thrombin to Cleave Protein C but Not Fibrinogen.

The W215A/E217A mutant thrombin is called "anticoagulant thrombin" because its activity toward its procoagulant substrate, fibrinogen, is reduced more than 500-fold whereas in the presence of thrombomodulin (TM) its activity toward its anticoagulant substrate, protein C, is reduced less than 10-fold. To understand how these mutations so dramatically alter one activity over the other, we compared the backbone dynamics of wild type thrombin to those of the W215A/E217A mutant thrombin by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS). Our results show that the mutations cause the 170s, 180s, and 220s C-terminal ß-barrel loops near the sites of mutation to exchange more, suggesting that the structure of this region is disrupted. Far from the mutation sites, residues at the N-terminus of the heavy chain, which need to be buried in the Ile pocket for correct structuring of the catalytic triad, also exchange much more than in wild type thrombin. TM binding causes reduced H/D exchange in these regions and also alters the dynamics of the ß-strand that links the TM binding site to the catalytic Asp 102 in both wild type thrombin and in the W215A/E217A mutant thrombin. In contrast, whereas TM binding reduces the dynamics the 170, 180 and 220 s C-terminal ß-barrel loops in WT thrombin, this region remains disordered in the W215A/E217A mutant thrombin. Thus, TM partially restores the catalytic activity of W215A/E217A mutant thrombin by allosterically altering its dynamics in a manner similar to that of wild type thrombin.

Development of a Rapid and Sensitive CasRx-Based Diagnostic Assay for SARS-CoV-2.

The development of an extensive toolkit for potential point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect SARS-CoV-2. Herein, we outline the development of an alternative CRISPR nucleic acid diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens XPD3002 (CasRx) to detect SARS-CoV-2, an approach we term SENSR (sensitive enzymatic nucleic acid sequence reporter) that can detect attomolar concentrations of SARS-CoV-2. We demonstrate 100% sensitivity in patient-derived samples by lateral flow and fluorescence readout with a detection limit of 45 copy/µL. This technology expands the available nucleic acid diagnostic toolkit, which can be adapted to combat future pandemics.

Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation.

Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-κB from DNA is achieved through accelerated dissociation by the inhibitor protein IκBα. Using single-molecule FRET, we observed a continuum of conformations of NF-κB in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-κB is partially bound, allowing IκBα invasion to facilitate DNA dissociation. IκBα induces a locked conformation where the DNA-binding domains of NF-κB are too far apart to bind DNA, whereas a loss-of-function IκBα mutant retains the NF-κB conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.