Discovery Of Inactive E* Conformations In Thrombin And Other Vitamin K- Dependent Clotting Proteases

Serine proteases of the chymotrypsin family play important roles in the regulation and function of numerous biological processes including digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. For many of these proteases, activity unfolds when a zymogen is activated by limited proteolysis and the associated conformational changes result in the formation of a proper active site and oxyanion hole, both of which are required for efficient hydrolysis of peptide bonds. The transition from zymogen to active enzyme, E, thus provides critical temporal and spatial regulatory mechanism of protease function. Catalytic activity of serine proteases belonging to Vitamin K-dependent clotting factors is significantly affected by Na+ through an allosteric mechanism. Over the past 30 years, structural and biochemical studies revealed that Na+ enhances the enzymatic properties of these proteases from a low activity, E, to a high activity: E:Na+) conformation. However, investigation of the effects of Na+ on these proteases has mainly focused on the thermodynamics of interaction and the resulting catalytic enhancement, with little emphasis on characterizing the kinetics of Na+ binding. In deed, the kinetic mechanism of Na+ binding to many Na+-activated enzymes remain for the most part unexplored due to lack of convenient probes to monitor the interaction or the difficulty of resolving rate constants for reactions that likely occur on a very fast time scale. My thesis project aims to fill this gap in the investigation of Na+-activated proteases by elucidating the kinetic mechanism of Na+ binding to vitamin K-dependent clotting factors. While studying the kinetics of Na+ binding to human α-thrombin, we observed a biphasic mechanism of binding whose analysis led to the discovery that in the absence of Na+, the enzyme exists in dynamic equilibrium between two conformations, E* and E. Structural and kinetic studies indicate that E is the active form of the enzyme responsible for its catalytic properties while E* is an inactive conformation that features a collapsed active site cleft, a disrupted oxyanion hole and an abrogated Na+ binding site. E* is not unique to α-thrombin, however, as we have observed a similar E* to E transition in meizo-thrombin-des F1, factor IXa, factor Xa and activated protein C. Discovery of E* to E transition embedded in these trypsin-like enzymes is novel, and the observation of E*-like features in structures of other serine proteases reveal a level of unprecedented conformational plasticity present in the chymotrypsin fold. The inter-conversion between E* and E has mechanistic significance on how these proteases function in vivo. Based on the physiological role of each protease, catalytic activity can be regulated by properly setting the E*-E equilibrium, favoring E* or E depending on whether that protease requires low or high catalytic activity for its in vivo function. More importantly, stabilization of E* through mutagenesis can provide a low activity enzyme incapable of interacting with substrate or binding inhibitor until an appropriate cofactor binds and unleashes its full catalytic activity. Using α &ndash thrombin, a key enzyme of blood coagulation as a model system, we demonstrated how each conformation could be stabilized through rational protein engineering using site-directed mutagenesis. Stabilization of its E* form will turn α-thrombin into an effective anticoagulant agent that can be utilized for in vivo therapeutic purposes. In fact, α-thrombin mutants, E217K and W215A/E217A that show anticoagulant and antithrombotic effects in non-human primates both exhibit some structural features of E* like partial collapse of the 215-217 β-strand and disruption of the oxyanion hole. Thus stabilization of E* through mutagenesis or binding of a small molecule can provide an elegant regulatory control that can fine tune specificity along a particular pathway. In addition, discovery of E* in Na+-activated clotting proteases expands our understanding of allostery in monomeric enzymes in general and in particular explains why the activity of some thrombin mutants is orders of magnitude lower than the activity of the wild-type in the absence of Na+. Findings from this thesis project reveal a fundamental property of structure-function regulation in the vitamin K dependent clotting enzymes and thus set the stage for further investigation of inactive conformations in other serine proteases of the chymotrypsin family. Whether the presence of E* is a universal property of all serine proteases will await future studies

A Tale of Loops and Tails: The Role of Intrinsically Disordered Protein Regions in R-Loop Recognition and Phase Separation

R-loops are non-canonical, three-stranded nucleic acid structures composed of a DNA:RNA hybrid, a displaced single-stranded (ss)DNA, and a trailing ssRNA overhang. R-loops perform critical biological functions under both normal and disease conditions. To elucidate their cellular functions, we need to understand the mechanisms underlying R-loop formation, recognition, signaling, and resolution. Previous high-throughput screens identified multiple proteins that bind R-loops, with many of these proteins containing folded nucleic acid processing and binding domains that prevent (e.g., topoisomerases), resolve (e.g., helicases, nucleases), or recognize (e.g., KH, RRMs) R-loops. However, a significant number of these R-loop interacting Enzyme and Reader proteins also contain long stretches of intrinsically disordered regions (IDRs). The precise molecular and structural mechanisms by which the folded domains and IDRs synergize to recognize and process R-loops or modulate R-loop-mediated signaling have not been fully explored. While studying one such modular R-loop Reader, the Fragile X Protein (FMRP), we unexpectedly discovered that the C-terminal IDR (C-IDR) of FMRP is the predominant R-loop binding site, with the three N-terminal KH domains recognizing the trailing ssRNA overhang. Interestingly, the C-IDR of FMRP has recently been shown to undergo spontaneous Liquid-Liquid Phase Separation (LLPS) assembly by itself or in complex with another non-canonical nucleic acid structure, RNA G-quadruplex. Furthermore, we have recently shown that FMRP can suppress persistent R-loops that form during transcription, a process that is also enhanced by LLPS via the assembly of membraneless transcription factories. These exciting findings prompted us to explore the role of IDRs in R-loop processing and signaling proteins through a comprehensive bioinformatics and computational biology study. Here, we evaluated IDR prevalence, sequence composition and LLPS propensity for the known R-loop interactome. We observed that, like FMRP, the majority of the R-loop interactome, especially Readers, contains long IDRs that are highly enriched in low complexity sequences with biased amino acid composition, suggesting that these IDRs could directly interact with R-loops, rather than being “mere flexible linkers” connecting the “functional folded enzyme or binding domains”. Furthermore, our analysis shows that several proteins in the R-loop interactome are either predicted to or have been experimentally demonstrated to undergo LLPS or are known to be associated with phase separated membraneless organelles. Thus, our overall results present a thought-provoking hypothesis that IDRs in the R-loop interactome can provide a functional link between R-loop recognition via direct binding and downstream signaling through the assembly of LLPS-mediated membrane-less R-loop foci. The absence or dysregulation of the function of IDR-enriched R-loop interactors can potentially lead to severe genomic defects, such as the widespread R-loop-mediated DNA double strand breaks that we recently observed in Fragile X patient-derived cells

Measuring Solvent Hydrogen Exchange Rates by Multifrequency Excitation 15 N CEST: Application to Protein Phase Separation

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Targeting SLP76:ITK interaction separates GVHD from GVL in allo-HSCT

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematological malignancies, due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also mediated by these cells. Here, we assessed the effect of attenuating TCR-mediated SLP76:ITK interaction in GVL vs. GVHD effects after allo-HSCT. CD8 and CD4 donor T cells from mice expressing a Y145F mutation in SLP-76 did not cause GVHD but preserved GVL effects against B-ALL cells. SLP76Y145FKI CD8 and CD4 donor T cells also showed less inflammatory cytokine production and migration to GVHD target organs. We developed a novel peptide to specifically inhibit SLP76:ITK interactions, resulting in decreased phosphorylation of PLCγ1 and ERK, decreased cytokine production in human T cells, and separation of GVHD from GVL effects. Altogether, our data suggest that inhibiting SLP76:ITK interaction could be a therapeutic strategy to separate GVHD from GVL effects after allo-HSCT treatment

Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) Differentiates GVL and GVHD in Allo-HSCT

Allogeneic hematopoietic stem cell transplantation is a potentially curative procedure for many malignant diseases. Donor T cells prevent disease recurrence graft-versus-leukemia (GVL) effect. Donor T cells also contribute to graft-versus-host disease (GVHD), a debilitating and potentially fatal complication. Novel treatment strategies are needed which allow preservation of GVL effects without causing GVHD. Using murine models, we show that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving GVL effects. Both CD8 and CD4 donor T cells from mice produce less inflammatory cytokines and show decrease migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). T cells exhibit reduced expression of IRF4 and decreased JAK/STAT signaling activity but upregulating expression of Eomesodermin (Eomes) and preserve cytotoxicity, necessary for GVL effect. Transcriptome analysis indicates that ITK signaling controls chemokine receptor expression during alloactivation, which in turn affects the ability of donor T cells to migrate to GVHD target organs. Our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment

Non-cooperative 4E-BP2 folding with exchange between eIF4E-binding and binding-incompatible states tunes cap-dependent translation inhibition

Phosphorylation of eIF4E binding proteins (4E-BPs) controls their folding and regulates cap-dependent translation. Here, the authors show that phosphorylation of the C-terminal disordered region stabilizes the non-cooperatively folded 4E-BP domain to an eIF4E binding-incompatible state to control translation

BME Weights for Non-Phosphorylated and 5-Phosphorylated 4E-BP2 ensembles generated with FastFloppyTail Deposited on the PED

Bayesian Maximum Entropy (BME) weights for NP- and 5P-4E-BP2 ensembles deposited on the Protein Ensemble Database (PED). 5p_100* correspond to weights for the N = 100 5-phosphorylated conformer ensemble, np_1000* correspond to weights for the N = 1000 non-phosphorylated conformer ensemble respectively