Impact of Nonnative Interactions on the Binding Kinetics of Intrinsically Disordered p53 with MDM2: Insights from All-Atom Simulation and Markov State Model Analysis.

Intrinsically disordered proteins (IDPs) lack a well-defined tertiary structure but are essential players in various biological processes. Their ability to undergo a disorder-to-order transition upon binding to their partners, known as the folding-upon-binding process, is crucial for their function. One classical example is the intrinsically disordered transactivation domain (TAD) of the tumor suppressor protein p53, which quickly forms a structured α-helix after binding to its partner MDM2, with clinical significance for cancer treatment. However, the contribution of nonnative interactions between the IDP and its partner to the rapid binding kinetics, as well as their interplay with native interactions, is not well understood at the atomic level. Here, we used molecular dynamics simulation and Markov state model (MSM) analysis to study the folding-upon-binding mechanism between p53-TAD and MDM2. Our results suggest that the system progresses from the nascent encounter complex to the well-structured encounter complex and finally reaches the native complex, following an induced-fit mechanism. We found that nonnative hydrophobic and hydrogen bond interactions, combined with native interactions, effectively stabilize the nascent and well-structured encounter complexes. Among the nonnative interactions, Leu25p53-Leu54MDM2 and Leu25p53-Phe55MDM2 are particularly noteworthy, as their interaction strength is close to the optimum. Evidently, strengthening or weakening these interactions could both adversely affect the binding kinetics. Overall, our findings suggest that nonnative interactions are evolutionarily optimized to accelerate the binding kinetics of IDPs in conjunction with native interactions.

Manure replacing synthetic fertilizer improves crop yield sustainability and reduces carbon footprint under winter wheat-summer maize cropping system.

Manure replacing synthetic fertilizer is a viable practice to ensure crop yield and increase soil organic carbon (SOC), but its impact on greenhouse gas (GHG) emissions is inconsistent, thus remains its effect on CF unclear. In this study, a 7-year field experiment was conducted to assess the impact of replacing synthetic fertilizer with manure on crop productivity, SOC sequestration, GHG emissions and crop CF under winter wheat-summer maize cropping system. Five treatments were involved: synthetic nitrogen, phosphorus, and potassium fertilizer (NPK) and 25%, 50%, 75%, and 100% of manure replacing synthetic N (25%M, 50%M, 75%M, and 100%M). Compared with NPK treatment, 25%M and 50%M treatments maintained annual yield (winter wheat plus summer maize) and sustainable yield index (SYI), but 75%M and 100%M treatments significantly decreased annual yield, and 100%M treatment also significantly reduced annual SYI. The SOC content exhibited a significant increasing trend over years in all treatments. After 7 years, SOC storage in manure treatments increased by 3.06-11.82 Mg ha-1 relative to NPK treatment. Manure treatments reduced annual GHG emissions by 14%-60% over NPK treatment. The CF of the cropping system ranged from 0.16 to 0.39 kg CO2 eq kg-1 of grain without considering SOC sequestration, in which the CF of manure treatments lowered by 18%-58% relative to NPK treatment. When SOC sequestration was involved in, the CF varied from -0.39 to 0.37 kg CO2 eq kg-1 of grain, manure treatments significantly reduced the CF by 22%-208% over NPK treatment. It was concluded that replacing 50% of synthetic fertilizer with manure was a sound option for achieving high crop yield and SYI but low CF under the tested cropping system.

O-methyltransferase-like enzyme catalyzed diazo installation in polyketide biosynthesis.

Diazo compounds are rare natural products possessing various biological activities. Kinamycin and lomaiviticin, two diazo natural products featured by the diazobenzofluorene core, exhibit exceptional potency as chemotherapeutic agents. Despite the extensive studies on their biosynthetic gene clusters and the assembly of their polyketide scaffolds, the formation of the characteristic diazo group remains elusive. L-Glutamylhydrazine was recently shown to be the hydrazine donor in kinamycin biosynthesis, however, the mechanism for the installation of the hydrazine group onto the kinamycin scaffold is still unclear. Here we describe an O-methyltransferase-like protein, AlpH, which is responsible for the hydrazine incorporation in kinamycin biosynthesis. AlpH catalyses a unique SAM-independent coupling of L-glutamylhydrazine and polyketide intermediate via a rare Mannich reaction in polyketide biosynthesis. Our discovery expands the catalytic diversity of O-methyltransferase-like enzymes and lays a strong foundation for the discovery and development of novel diazo natural products through genome mining and synthetic biology.

Combined prediction and design reveals the target recognition mechanism of an intrinsically disordered protein interaction domain.

An increasing number of protein interaction domains and their targets are being found to be intrinsically disordered proteins (IDPs). The corresponding target recognition mechanisms are mostly elusive because of challenges in performing detailed structural analysis of highly dynamic IDP-IDP complexes. Here, we show that by combining recently developed computational approaches with experiments, the structure of the complex between the intrinsically disordered C-terminal domain (CTD) of protein 4.1G and its target IDP region in NuMA can be dissected at high resolution. First, we carry out systematic mutational scanning using dihydrofolate reductase-based protein complementarity analysis to identify essential interaction regions and key residues. The results are found to be highly consistent with an α/ß-type complex structure predicted by AlphaFold2 (AF2). We then design mutants based on the predicted structure using a deep learning protein sequence design method. The solved crystal structure of one mutant presents the same core structure as predicted by AF2. Further computational prediction and experimental assessment indicate that the well-defined core structure is conserved across complexes of 4.1G CTD with other potential targets. Thus, we reveal that an intrinsically disordered protein interaction domain uses an α/ß-type structure module formed through synergistic folding to recognize broad IDP targets. Moreover, we show that computational prediction and experiment can be jointly applied to segregate true IDP regions from the core structural domains of IDP-IDP complexes and to uncover the structure-dependent mechanisms of some otherwise elusive IDP-IDP interactions.

Different conformational dynamics of SNARE protein Ykt6 among yeast and mammals.

Ykt6 is one of the most conserved SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) proteins involved in multiple intracellular membrane trafficking processes. The membrane-anchoring function of Ykt6 has been elucidated to result from its conformational transition from a closed state to an open state. Two ways of regulating the conformational transition were proposed: the C-terminal lipidation and the phosphorylation at the SNARE core. Despite many aspects of common properties, Ykt6 displays differential cellular localizations and functional behaviors in different species, such as yeast, mammals, and worms. The structure-function relationship underlying these differences remains elusive. Here, we combined biochemical characterization, single-molecule FRET measurement, and molecular dynamics simulation to compare the conformational dynamics of yeast and rat Ykt6. Compared to rat Ykt6 (rYkt6), yeast Ykt6 (yYkt6) has more open conformations and could not bind dodecylphosphocholine that inhibits rYkt6 in the closed state. A point mutation T46L/Q57A was shown to be able to convert yYkt6 to a more closed and dodecylphosphocholine-bound state, where Leu46 contributes key hydrophobic interactions for the closed state. We also demonstrated that the phospho-mutation S174D could shift the conformation of rYkt6 to a more open state, but the corresponding mutation S176D in yYkt6 leads to a slightly more closed conformation. These observations shed light on the regulatory mechanism underlying the variations of Ykt6 functions across species.

Fast Discrimination of Sialylated N-Glycan Linkage Isomers with One-Step Derivatization by Microfluidic Capillary Electrophoresis-Mass Spectrometry.

Linkage isomers (α-2,3- or α-2,6-linkage) of sialylated N-glycans are involved in the emergence and progression of some diseases, so they are of great significance for diagnosing and monitoring diseases. However, the qualitative and quantitative analysis of sialylated N-glycan linkage isomers remains challenging due to their low abundance and limited isomeric separation techniques. Herein, we developed a novel strategy integrating one-step sialic acid derivatization, positive charge-sensitive separation and highly sensitive detection based on microfluidic capillary electrophoresis-mass spectrometry (MCE-MS) for fast and specific analysis of α-2,3- and α-2,6-linked sialylated N-glycan isomers. A kind of easily charged long-chain amino compound was screened first for one-step sialic acid derivatization so that only α-2,3- and α-2,6-linked isomers can be quickly and efficiently separated within 10 min by MCE due to the difference in structural conformation, whose separation mechanism was further theoretically supported by molecular dynamic simulation. In addition, different sialylated N-glycans were separated in order according to the number of sialic acids, so that a migration time-based prediction of the number of sialic acids was achieved. Finally, the sialylated N-glycome of human serum was profiled within 10 min and 6 of the 52 detected sialylated N-glycans could be potential diagnostic biomarkers of cervical cancer (CC), whose α-2,3- and α-2,6-linked isomers were distinguished by α-2,3Neuraminidase S.

The importance of the compact disordered state in the fuzzy interactions between intrinsically disordered proteins.

The intrinsically disordered C-terminal domain (CTD) of protein 4.1G is able to specifically bind a 26-residue intrinsically disordered region of NuMA, forming a dynamic fuzzy complex. As one of a few cases of extremely fuzzy interactions between two intrinsically disordered proteins/regions (IDPs/IDRs) without induced folding, the principle of the binding is unknown. Here, we combined experimental and computational methods to explore the detailed mechanism of the interaction between 4.1G-CTD and NuMA. MD simulations suggest that the kinetic hub states in the structure ensemble of 4.1G-CTD are favorable in the fuzzy complex. The feature of these hub states is that the binding 'hot spot' motifs ßA and ßB exhibit ß strand propensities and are well packed to each other. The binding between 4.1G-CTD and NuMA is disrupted at low pH, which changes the intramolecular packing of 4.1G-CTD and weakens the packing between ßA and ßB motifs. Low pH conditions also lead to increased hydrodynamic radius and acceleration of backbone dynamics of 4.1G-CTD. All these results underscore the importance of tertiary structural arrangements and overall compactness of 4.1G-CTD in its binding to NuMA, i.e. the compact disordered state of 4.1G-CTD is crucial for binding. Different from the short linear motifs (SLiMs) that are often found to mediate IDP interactions, 4.1G-CTD functions as an intrinsically disordered domain (IDD), which is a functional and structural unit similar to conventional protein domains. This work sheds light on the molecular recognition mechanism of IDPs/IDRs and expands the conventional structure-function paradigm in protein biochemistry.

Unexpected Role of a Short-Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis.

We investigated the biosynthetic pathway of type II polyketide murayaquinone. The murayaquinone biosynthetic cluster contains genes for three putative short-chain dehydrogenase/reductase family enzymes including MrqF and MrqH with known functions and MrqM with unclear function. We report the functional characterization of MrqM for its role in murayaquinone biosynthesis. Our gene deletion experiment and structural elucidation of the accumulated intermediates revealed that MrqM is related with the second polyketide ring cyclization, because the inactivation of mrqM resulted in the accumulation of an off-pathway intermediate SEK43 and disrupted the second and third ring cyclization. Site-directed mutagenesis studies showed that two conserved residues in MrqM and homologous proteins Y151 and K155 are essential for its activity. The previously proposed second/third ring cyclase, MrqD, might instead play another important role in the chain releasing step of the murayaquinone biosynthesis.

Genome-Wide Analysis of the YABBY Transcription Factor Family in Rapeseed (Brassica napus L.).

The YABBY family of plant-specific transcription factors play important regulatory roles during the development of leaves and floral organs, but their functions in Brassica species are incompletely understood. Here, we identified 79 YABBY genes from Arabidopsis thaliana and five Brassica species (B. rapa, B. nigra, B. oleracea, B. juncea, and B. napus). A phylogenetic analysis of YABBY proteins separated them into five clusters (YAB1-YAB5) with representatives from all five Brassica species, suggesting a high degree of conservation and similar functions within each subfamily. We determined the gene structure, chromosomal location, and expression patterns of the 21 BnaYAB genes identified, revealing extensive duplication events and gene loss following polyploidization. Changes in exon-intron structure during evolution may have driven differentiation in expression patterns and functions, combined with purifying selection, as evidenced by Ka/Ks values below 1. Based on transcriptome sequencing data, we selected nine genes with high expression at the flowering stage. qRT-PCR analysis further indicated that most BnaYAB family members are tissue-specific and exhibit different expression patterns in various tissues and organs of B. napus. This preliminary study of the characteristics of the YABBY gene family in the Brassica napus genome provides theoretical support and reference for the later functional identification of the family genes.

Voltage-Driven Flipping of Zwitterionic Artificial Channels in Lipid Bilayers to Rectify Ion Transport.

We developed a voltage-sensitive artificial transmembrane channel by mimicking the dipolar structure of natural alamethicin channel. The artificial channel featured a zwitterionic structure and could undergo voltage-driven flipping in the lipid bilayers. Importantly, this flipping of the channel could lead to their directional alignment in the bilayers and rectifying behavior for ion transport.

The Role of Calcium in Regulating the Conformational Dynamics of d-Galactose/d-Glucose-Binding Protein Revealed by Markov State Model Analysis.

The d-glucose/d-galactose-binding protein (GGBP) from Escherichia coli is a substrate-binding protein (SBP) associated with sugar transport and chemotaxis. It is also a calcium-binding protein, which makes it unique in the SBP family. However, the functional importance of Ca2+ binding is not fully understood. Here, the calcium-dependent properties of GGBP were explored by all-atom molecular dynamics simulations and Markov state model (MSM) analysis as well as single-molecule Förster resonance energy transfer (smFRET) measurements. In agreement with previous experimental studies, we observed the structure stabilization effect of Ca2+ binding on the C-terminal domain of GGBP, especially the Ca2+-binding site. Interestingly, the MSMs of calcium-depleted GGBP and calcium-bound GGBP (GGBP/Ca2+) demonstrate that Ca2+ greatly stabilizes the open conformation, and smFRET measurements confirmed this result. Further analysis reveals that Ca2+ binding disturbs the local hydrogen bonding interactions and the conformational dynamics of the hinge region, thereby weakening the long-range interdomain correlations to favor the open conformation. These results suggest an active regulatory role of Ca2+ binding in GGBP, which finely tunes the conformational distribution. The work sheds new light on the study of calcium-binding proteins in prokaryotes.

Recent advances in atomic molecular dynamics simulation of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) play important roles in cellular functions. The inherent structural heterogeneity of IDPs makes the high-resolution experimental characterization of IDPs extremely difficult. Molecular dynamics (MD) simulation could provide the atomic-level description of the structural and dynamic properties of IDPs. This perspective reviews the recent progress in atomic MD simulation studies of IDPs, including the development of force fields and sampling methods, as well as applications in IDP-involved protein-protein interactions. The employment of large-scale simulations and advanced sampling techniques allows more accurate estimation of the thermodynamics and kinetics of IDP-mediated protein interactions, and the holistic landscape of the binding process of IDPs is emerging.

Revealing Thermodynamics and Kinetics of Lipid Self-Assembly by Markov State Model Analysis.

Self-assembly is ubiquitous in the realm of biology and has become an elegant bottom-up approach to fabricate new materials. Although molecular dynamics (MD) simulations can complement experiments by providing the missing atomic details, it still remains a grand challenge to reveal the thermodynamic and kinetic information on a self-assembly system. In this work, we demonstrate for the first time that the Markov state model analysis can be used to delineate the variation of free energy during the self-assembly process of a typical amphiphilic lipid dipalmitoyl-phosphatidylcholine (DPPC). Free energy profiles against the solvent-accessible surface area and the root-mean-square deviation have been derived from extensive MD results of more than five hundred trajectories, which identified a metastable crossing-cylinder (CC) state and a transition state of the distorted bilayer with a free energy barrier of ∼0.02 kJ mol-1 per DPPC lipid, clarifying a long-standing speculation for 20 years that there exists a free energy barrier during lipid self-assembly. Our simulations also unearth two mesophase structures at the early stage of self-assembly, discovering two assembling pathways to the CC state that have never been reported before. Further thermodynamic analysis derives the contributions from the enthalpy and the entropy terms to the free energy, demonstrating the critical role played by the enthalpy-entropy compensation. Our strategy opens the door to quantitatively understand the self-assembly processes in general and provides new opportunities for identifying common thermodynamic and kinetic patterns in different self-assembly systems and inspiring new ideas for experiments. It may also contribute to the refinement of force field parameters of various self-assembly systems.

Artificial Aquaporin That Restores Wound Healing of Impaired Cells.

Artificial aquaporins are synthetic molecules that mimic the structure and function of natural aquaporins (AQPs) in cell membranes. The development of artificial aquaporins would provide an alternative strategy for treatment of AQP-related diseases. In this report, an artificial aquaporin has been constructed from an amino-terminated tubular molecule, which operates in a unimolecular mechanism. The artificial channel can work in cell membranes with high water permeability and selectivity rivaling those of AQPs. Importantly, the channel can restore wound healing of the cells that contain function-lost AQPs.

Ligand-bound glutamine binding protein assumes multiple metastable binding sites with different binding affinities.

Protein dynamics plays key roles in ligand binding. However, the microscopic description of conformational dynamics-coupled ligand binding remains a challenge. In this study, we integrate molecular dynamics simulations, Markov state model (MSM) analysis and experimental methods to characterize the conformational dynamics of ligand-bound glutamine binding protein (GlnBP). We show that ligand-bound GlnBP has high conformational flexibility and additional metastable binding sites, presenting a more complex energy landscape than the scenario in the absence of ligand. The diverse conformations of GlnBP demonstrate different binding affinities and entail complex transition kinetics, implicating a concerted ligand binding mechanism. Single molecule fluorescence resonance energy transfer measurements and mutagenesis experiments are performed to validate our MSM-derived structure ensemble as well as the binding mechanism. Collectively, our study provides deeper insights into the protein dynamics-coupled ligand binding, revealing an intricate regulatory network underlying the apparent binding affinity.

Author Correction: Structural and dynamic studies reveal that the Ala-rich region of ataxin-7 initiates α-helix formation of the polyQ tract but suppresses its aggregation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dynamic multivalent interactions of intrinsically disordered proteins.

Protein-protein interactions involving intrinsically disordered proteins (IDPs) usually display dynamic and multivalent features. Recent experimental data revealed myriad functional roles of the dynamic multivalent interaction (DMI) of IDPs. However, characterization of DMI remains a challenge due to its complex and promiscuous nature. Recent studies start showing that understanding the mechanistic role of DMI relies on combined use of various techniques and construction of microscopic models in elucidating the binding thermodynamics and kinetics.

Glycerol transport through the aquaglyceroporin GlpF: bridging dynamics and kinetics with atomic simulation.

The aquaglyceroporin GlpF is a member of the aquaporin family. It selectively conducts small molecules, such as glycerol, across the cell membrane under a concentration gradient of the substrate. Atomistic molecular dynamics (MD) simulation would provide great insight into the substrate transport mechanism of GlpF and membrane channels alike. Ideally, non-equilibrium simulations under various concentration gradients of glycerol are desired to emulate the transportation in cells, but this kind of simulation is difficult due to a complicated system setup and high computational cost. Here, we present a new strategy to extract non-equilibrium kinetic information from equilibrium MD simulation. We first performed long-time (totally 22.5 µs) multi-copy equilibrium MD simulations of glycerol conduction through GlpF. Tens of times the spontaneous permeation of glycerol through GlpF was observed, allowing us to elucidate the detailed mechanism of the stereoselectivity for glycerol. Then we employed Markov state model (MSM) analysis of the MD trajectories to identify the intermediate states during glycerol transport and calculate the inter-state transition rate constants. Based on the results of MSM analysis, we built the kinetic models of glycerol transport and calculated the glycerol fluxes under various concentration gradients by solving the master equations. The results agree well with the experimental measurement at a certain glycerol concentration, and provide holistic information on the glycerol conduction capacity of GlpF. Our work demonstrates that long-time atomistic MD simulations can now bridge the microscopic dynamics and the kinetic description of substance transport through membrane channels, hopefully facilitating the engineering of new selective channels for various molecules.

Facile Fabrication of Durable Superhydrophobic Films from Carbon Nanotube/Main-Chain Type Polybenzoxazine Composites.

Superhydrophobic materials have immense applications in the fields of industry and research. However, their durability is still a cause for concern. A facile method for preparing durable superhydrophobic films from carbon nanotubes (CNTs) and the main-chain type polybenzoxazine precursors is reported herein. We used probe ultrasonicator to prepare CNT/polybenzoxazine coatings. Compared with the general sonicating dispersion process, the dispersion time was greatly reduced from a few hours to 5 minutes and the prepared suspension exhibited film-forming characteristics well. The CNT/polybenzoxazine films, which do not contain any fluorinated compounds, exhibit remarkable durability against thermal treatment, organic solvents, corrosive liquids, and sandpaper abrasion, while retaining their superhydrophobicity. Furthermore, these CNT/polybenzoxazine films also showed durable superhydrophobicity after ultraviolet (UV) irradiation for 100 h. This CNT/polybenzoxazine film can be readily used for practical applications to make durable superhydrophobic coatings.