Structural basis for product specificities of MLL family methyltransferases.

Human mixed-lineage leukemia (MLL) family methyltransferases methylate histone H3 lysine 4 to different methylation states (me1/me2/me3) with distinct functional outputs, but the mechanism underlying the different product specificities of MLL proteins remains unclear. Here, we develop methodologies to quantitatively measure the methylation rate difference between mono-, di-, and tri-methylation steps and demonstrate that MLL proteins possess distinct product specificities in the context of the minimum MLL-RBBP5-ASH2L complex. Comparative structural analyses of MLL complexes by X-ray crystal structures, fluorine-19 nuclear magnetic resonance, and molecular dynamics simulations reveal that the dynamics of two conserved tyrosine residues at the "F/Y (phenylalanine/tyrosine) switch" positions fine-tune the product specificity. The variation in the intramolecular interaction between SET-N and SET-C affects the F/Y switch dynamics, thus determining the product specificities of MLL proteins. These results indicate a modified F/Y switch rule applicable for most SET domain methyltransferases and implicate the functional divergence of MLL proteins.

An enzyme-based biosensor for monitoring and engineering protein stability in vivo.

Protein stability affects the physiological functions of proteins and is also a desirable trait in many protein engineering tasks, yet improving protein stability is challenging because of limitations in methods for directly monitoring protein stability in cells. Here, we report an in vivo stability biosensor wherein a protein of interest (POI) is inserted into a microbial enzyme (CysGA) that catalyzes the formation of endogenous fluorescent compounds, thereby coupling POI stability to simple fluorescence readouts. We demonstrate the utility of the biosensor in directed evolution to obtain stabilized, less aggregation-prone variants of two POIs (including nonamyloidogenic variants of human islet amyloid polypeptide). Beyond engineering applications, we exploited our biosensor in deep mutational scanning for experimental delineation of the stability-related contributions of all residues throughout the catalytic domain of a histone H3K4 methyltransferase, thereby revealing its scientifically informative stability landscape. Thus, our highly accessible method for in vivo monitoring of the stability of diverse proteins will facilitate both basic research and applied protein engineering efforts.

Simultaneous Improvement in the Thermostability and Catalytic Activity of Epoxidase Lsd18 for the Synthesis of Lasalocid A.

Enzymes used in the synthesis of natural products are potent catalysts, capable of efficient and stereoselective chemical transformations. Lsd18 catalyzes two sequential epoxidations during the biosynthesis of lasalocid A, a polyether polyketide natural product. We performed protein engineering on Lsd18 to improve its thermostability and catalytic activity. Utilizing structure-guided methods of FoldX and Rosetta-ddG, we designed 15 mutants of Lsd18. Screening of these mutants using thermal shift assay identified stabilized variants Lsd18-T189M, Lsd18-S195M, and the double mutant Lsd18-T189M-S195M. Trypsin digestion, molecular dynamic simulation, circular dichroism (CD) spectroscopy, and X-ray crystallography provided insights into the molecular basis for the improved enzyme properties. Notably, enhanced hydrophobic interaction within the enzyme core and interaction of the protein with the FAD cofactor appear to be responsible for its better thermostability.

Distinct kinetic mechanisms of H3K4 methylation catalyzed by MLL3 and MLL4 core complexes.

The methyltransferases MLL3 and MLL4 primarily catalyze the monomethylation of histone H3 lysine 4 (H3K4) on enhancers to regulate cell-type-specific gene expression and cell fate transition. MLL3 and MLL4 share almost identical binding partners and biochemical activities, but perform specific and nonredundant functions. The features and functions that distinguish MLL3 and MLL4 remain elusive. Here, we characterize the kinetic mechanisms of MLL3 and MLL4 ternary complexes containing the catalytic SET domain from MLL3 or MLL4 (MLL3SET or MLL4SET), the SPRY domain of ASH2L (ASH2LSPRY), and a short fragment of RBBP5 (RBBP5AS-ABM) to search for possible explanations. Steady-state kinetic analyses and inhibition studies reveal that the MLL3 complex catalyzes methylation in a random sequential bi-bi mechanism. In contrast, the MLL4 complex adopts an ordered sequential bi-bi mechanism, in which the cofactor S-adenosylmethionine (AdoMet) binds to the enzyme prior to the H3 peptide, and the methylated H3 peptide dissociates from the enzyme before S-adenosylhomocysteine (AdoHcy) detaches after methylation. Substrate-binding assays using fluorescence polarization (FP) confirm that AdoMet binding is a prerequisite for H3 binding for the MLL4 complex but not for the MLL3 complex. Molecular dynamic simulations reveal that the binding of AdoMet exclusively induces conformational constraints on the AdoMet-binding groove and the H3 substrate-binding pocket of MLL4, therefore stabilizing a specific active conformation to ease entry of the substrate H3. The distinct kinetic mechanisms and conformational plasticities provide important insights into the differential functions of MLL3 and MLL4 and may also guide the development of selective inhibitors targeting MLL3 or MLL4.

Molecular Characterization of an Intrinsically Disordered Chaperone Reveals Net-Charge Regulation in Chaperone Action.

Molecular chaperones are diverse biomacromolecules involved in the maintenance of cellular protein homeostasis (proteostasis). Here we demonstrate that in contrast to most chaperones with defined three-dimensional structures, the acid-inducible protein Asr in Escherichia coli is intrinsically disordered and exhibits varied aggregation-preventing or aggregation-promoting activities, acting as a "conditionally active chaperone". Bioinformatics and experimental analyses of Asr showed that it is devoid of hydrophobic patches but rich in positive charges and local polyproline II backbone structures. Asr contributes to the integrity of the bacterial outer membrane under mildly acidic conditions in vivo and possesses chaperone activities toward model clients in vitro. Notably, its chaperone activity is dependent on the net charges of clients: on the one hand, it inhibits the aggregation of clients with similar net charges; on the other hand, it stimulates the aggregation of clients with opposite net charges. Mutational analysis confirmed that positively charged residues in Asr are essential for the varied effects on protein aggregation, suggesting that electrostatic interactions are the major driving forces underlying Asr's proteostasis-related activity. These findings present a unique example of an intrinsically disordered molecular chaperone with distinctive dual functions-as an aggregase or as a chaperone-depending on the net charges of clients.

Insights into the client protein release mechanism of the ATP-independent chaperone Spy.

Molecular chaperones play a central role in regulating protein homeostasis, and their active forms often contain intrinsically disordered regions (IDRs). However, how IDRs impact chaperone action remains poorly understood. Here, we discover that the disordered N terminus of the prototype chaperone Spy facilitates client release. With NMR spectroscopy and molecular dynamics simulations, we find that the N terminus can bind transiently to the client-binding cavity of Spy primarily through electrostatic interactions mediated by the N-terminal D26 residue. This intramolecular interaction results in a dynamic competition of the N terminus with the client for binding to Spy, which promotes client discharge. Our results reveal the mechanism by which Spy releases clients independent of energy input, thus enriching the current knowledge on how ATP-independent chaperones release their clients and highlighting the importance of synergy between IDRs and structural domains in regulating protein function.

DPY30 acts as an ASH2L-specific stabilizer to stimulate the enzyme activity of MLL family methyltransferases on different substrates.

Dumpy-30 (DPY30) is a conserved component of the mixed lineage leukemia (MLL) family complex and is essential for robust methyltransferase activity of MLL complexes. However, the biochemical role of DPY30 in stimulating methyltransferase activity of MLL complexes remains elusive. Here, we demonstrate that DPY30 plays a crucial role in regulating MLL1 activity through two complementary mechanisms: A nucleosome-independent mechanism and a nucleosome-specific mechanism. DPY30 functions as an ASH2L-specific stabilizer to increase the stability of ASH2L and enhance ASH2L-mediated interactions. As a result, DPY30 promotes the compaction and stabilization of the MLL1 complex, consequently increasing the HKMT activity of the MLL1 complex on diverse substrates. DPY30-stabilized ASH2L further acquires additional interfaces with H3 and nucleosomal DNA, thereby boosting the methyltransferase activity of the MLL1 complex on nucleosomes. These results collectively highlight the crucial and conserved roles of DPY30 in the complex assembly and activity regulation of MLL family complexes.

Online bioinformatics teaching practice: Comparison of popular docking programs using SARS-CoV-2 spike RBD-ACE2 complex as a benchmark.

In this information era, there is an urgent need for tighter integration of bioinformatics and experimental biology. The enormous amount of data generated by biological experiments calls for extensive computational analysis. Many bioinformatics textbooks at present mainly focus on theories, which hinders the vigorous development of scientific research. As a result, most students are simply familiar with the bioinformatics theories but lack the opportunity to put them into practice. Here, we present our bioinformatics docking project conducted during the self-isolation period of the COVID-19 pandemic. Five students used the RBD-ACE2 complex as a benchmark to conduct a systematic comparison of several open-source online molecular docking programs. The virus surface spike protein mediates the entry of the SARS-CoV-2 virus into human cells by binding to its receptor, angiotensin-converting enzyme 2 (ACE2), through its receptor-binding domain (RBD). Through docking and comparing predicted structures to the crystal structure, students gained the opportunity to practice different bioinformatics tools independently and conduct research collaboratively. It opens a window for students to reach out to the state-of-the-art bioinformatics techniques and to keep up with the research trends. The online workshop has also proven to be an innovative method for bioinformatics teaching. We hope our work can inspire other educators to develop strategies to expose undergraduate students to modern bioinformatics and turn every temporary difficulty into a possible learning opportunity.