Three-metal ion mechanism of cross-linked and uncross-linked DNA polymerase ß: A theoretical study.

In our recent publication, we have proposed a revised base excision repair pathway in which DNA polymerase ß (Polß) catalyzes Schiff base formation prior to the gap-filling DNA synthesis followed by ß-elimination. In addition, the polymerase activity of Polß employs the "three-metal ion mechanism" instead of the long-standing "two-metal ion mechanism" to catalyze phosphodiester bond formation based on the fact derived from time-resolved x-ray crystallography that a third Mg2+ was captured in the polymerase active site after the chemical reaction was initiated. In this study, we develop the models of the uncross-linked and cross-linked Polß complexes and investigate the "three-metal ion mechanism" vs the "two-metal ion mechanism" by using the quantum mechanics/molecular mechanics molecular dynamics simulations. Our results suggest that the presence of the third Mg2+ ion stabilizes the reaction-state structures, strengthens correct nucleotide binding, and accelerates phosphodiester bond formation. The improved understanding of Polß's catalytic mechanism provides valuable insights into DNA replication and damage repair.

Investigations of the underlying mechanisms of HIF-1α and CITED2 binding to TAZ1.

The TAZ1 domain of CREB binding protein is crucial for transcriptional regulation and recognizes multiple targets. The interactions between TAZ1 and its specific targets are related to the cellular hypoxic negative feedback regulation. Previous experiments reported that one of the TAZ1 targets, CITED2, is an efficient competitor of another target, HIF-1α. Here, by developing the structure-based models of TAZ1 complexes, we have uncovered the underlying mechanisms of the competitions between the two intrinsic disordered proteins (IDPs) HIF-1α and CITED2 binding to TAZ1. Our results support the experimental hypothesis on the competition mechanisms and the apparent affinity. Furthermore, the simulations locate the dominant position of forming TAZ1-CITED2 complex in both thermodynamics and kinetics. For thermodynamics, TAZ1-CITED2 is the lowest basin located on the free energy surface of binding in the ternary system. For kinetics, the results suggest that CITED2 binds to TAZ1 faster than HIF-1α. In addition, the analysis of contact map and Φ values is important for guiding further experimental studies to understand the biomolecular functions of IDPs.

Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble.

Liquid-liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.

Influence of sequence length and charged residues on Swc5 binding with histone H2A-H2B.

SWR is a member of chromatin remodeler family and participates the replacement of histone H2A with H2A.Z. One of the SWR subunits, Swc5, has an intrinsically disordered region and binds to H2A-H2B dimer. Though the binding structure of Swc5 and H2A-H2B has been resolved recently, it is still challenging to investigate the binding mechanism as well as the role of the charge interactions between Swc5 and H2A-H2B. Here we developed a coarse-grained structure-based model and performed molecular dynamics simulations to investigate the binding processes of two Swc5 regions with different lengths (swc5-a and swc5-b) to H2A-H2B. The simulation results suggest a different role of electrostatic interactions between swc5-a/swc5-b and H2A-H2B on binding. The electrostatic interactions between swc5-a/swc5-b and H2A-H2B can not only accelerate the initial capture step of binding, but can also trap the swc5-a/swc5-b at the wrong binding site on H2A. Besides, the conserved DEF/Y-2 motif of Swc5 is important for the binding affinity and the recognition with H2A-H2B at the initial step. Both swc5-a and swc5-b undergo a structural shift before reaching the final bound state. This theoretical study provides important details and the underlying physical mechanisms of the binding processes of swc5-a/swc5-b and H2A-H2B.

Physics of biomolecular recognition and conformational dynamics.

Biomolecular recognition usually leads to the formation of binding complexes, often accompanied by large-scale conformational changes. This process is fundamental to biological functions at the molecular and cellular levels. Uncovering the physical mechanisms of biomolecular recognition and quantifying the key biomolecular interactions are vital to understand these functions. The recently developed energy landscape theory has been successful in quantifying recognition processes and revealing the underlying mechanisms. Recent studies have shown that in addition to affinity, specificity is also crucial for biomolecular recognition. The proposed physical concept of intrinsic specificity based on the underlying energy landscape theory provides a practical way to quantify the specificity. Optimization of affinity and specificity can be adopted as a principle to guide the evolution and design of molecular recognition. This approach can also be used in practice for drug discovery using multidimensional screening to identify lead compounds. The energy landscape topography of molecular recognition is important for revealing the underlying flexible binding or binding-folding mechanisms. In this review, we first introduce the energy landscape theory for molecular recognition and then address four critical issues related to biomolecular recognition and conformational dynamics: (1) specificity quantification of molecular recognition; (2) evolution and design in molecular recognition; (3) flexible molecular recognition; (4) chromosome structural dynamics. The results described here and the discussions of the insights gained from the energy landscape topography can provide valuable guidance for further computational and experimental investigations of biomolecular recognition and conformational dynamics.

Investigation of sliding DNA clamp dynamics by single-molecule fluorescence, mass spectrometry and structure-based modeling.

Proliferating cell nuclear antigen (PCNA) is a trimeric ring-shaped clamp protein that encircles DNA and interacts with many proteins involved in DNA replication and repair. Despite extensive structural work to characterize the monomeric, dimeric, and trimeric forms of PCNA alone and in complex with interacting proteins, no structure of PCNA in a ring-open conformation has been published. Here, we use a multidisciplinary approach, including single-molecule Förster resonance energy transfer (smFRET), native ion mobility-mass spectrometry (IM-MS), and structure-based computational modeling, to explore the conformational dynamics of a model PCNA from Sulfolobus solfataricus (Sso), an archaeon. We found that Sso PCNA samples ring-open and ring-closed conformations even in the absence of its clamp loader complex, replication factor C, and transition to the ring-open conformation is modulated by the ionic strength of the solution. The IM-MS results corroborate the smFRET findings suggesting that PCNA dynamics are maintained in the gas phase and further establishing IM-MS as a reliable strategy to investigate macromolecular motions. Our molecular dynamic simulations agree with the experimental data and reveal that ring-open PCNA often adopts an out-of-plane left-hand geometry. Collectively, these results implore future studies to define the roles of PCNA dynamics in DNA loading and other PCNA-mediated interactions.

Binding mechanism and dynamic conformational change of C subunit of PKA with different pathways.

The catalytic subunit of PKA (PKAc) exhibits three major conformational states (open, intermediate, and closed) during the biocatalysis process. Both ATP and substrate/inhibitor can effectively induce the conformational changes of PKAc from open to closed states. Aiming to explore the mechanism of this allosteric regulation, we developed a coarse-grained model and analyzed the dynamics of conformational changes of PKAc during binding by performing molecular dynamics simulations for apo PKAc, binary PKAc (PKAc with ATP, PKAc with PKI), and ternary PKAc (PKAc with ATP and PKI). Our results suggest a mixed binding mechanism of induced fit and conformational selection, with the induced fit dominant. The ligands can drive the movements of Gly-rich loop as well as some regions distal to the active site in PKAc and stabilize them at complex state. In addition, there are two parallel pathways (pathway with PKAc-ATP as an intermediate and pathway PKAc-PKI as an intermediate) during the transition from open to closed states. By molecular dynamics simulations and rate constant analyses, we find that the pathway through PKAc-ATP intermediate is the main binding route from open to closed state because of the fact that the bound PKI will hamper ATP from successful binding and significantly increase the barrier for the second binding subprocess. These findings will provide fundamental insights of the mechanisms of PKAc conformational change upon binding.

Role of non-native electrostatic interactions in the coupled folding and binding of PUMA with Mcl-1.

PUMA, which belongs to the BH3-only protein family, is an intrinsically disordered protein (IDP). It binds to its cellular partner Mcl-1 through its BH3 motif, which folds upon binding into an α helix. We have applied a structure-based coarse-grained model, with an explicit Debye-Hückel charge model, to probe the importance of electrostatic interactions both in the early and the later stages of this model coupled folding and binding process. This model was carefully calibrated with the experimental data on helical content and affinity, and shown to be consistent with previously published experimental data on binding rate changes with respect to ionic strength. We find that intramolecular electrostatic interactions influence the unbound states of PUMA only marginally. Our results further suggest that intermolecular electrostatic interactions, and in particular non-native electrostatic interactions, are involved in formation of the initial encounter complex. We are able to reveal the binding mechanism in more detail than is possible using experimental data alone however, and in particular we uncover the role of non-native electrostatic interactions. We highlight the potential importance of such electrostatic interactions for describing the binding reactions of IDPs. Such approaches could be used to provide predictions for the results of mutational studies.

Enzymatic Activity and Thermodynamic Stability of Biliverdin IXß Reductase Are Maintained by an Active Site Serine.

Biliverdin reductase IXß (BLVRB) is a crucial enzyme in heme metabolism. Recent studies in humans have identified a loss-of-function mutation (Ser111Leu) that unmasks a fundamentally important role in hematopoiesis. We have undertaken experimental and thermodynamic modeling studies to provide further insight into the role of the cofactor in substrate accessibility and protein folding properties regulating BLVRB catalytic mechanisms. Site-directed mutagenesis with molecular dynamic (MD) simulations establish the critical role of NAD(P)H-dependent conformational changes on substrate accessibility by forming the "hydrophobic pocket", along with identification of a single key residue (Arg35) modulating NADPH/NADH selectivity. Loop80 and Loop120 block the hydrophobic substrate binding pocket in apo BLVRB (open), whereas movement of these structures after cofactor binding results in the "closed" (catalytically active) conformation. Both enzymatic activity and thermodynamic stability are affected by mutation(s) involving Ser111, which is located in the core of the BLVRB active site. This work 1) elucidates the crucial role of Ser111 in enzymatic catalysis and thermodynamic stability by active site hydrogen bond network; 2) defines a dynamic model for apo BLVRB extending beyond the crystal structure of the binary BLVRB/NADP+ complex; 3) provides a structural basis for the "encounter" and "equilibrium" states of the binary complex, which are regulated by NAD(P)H.

Insights into the phosphatase and the synthase activities of human bisphosphoglycerate mutase: a quantum mechanics/molecular mechanics simulation.

Bisphosphoglycerate mutase (BPGM) is a multi-activity enzyme. Its main function is to synthesize the 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. This enzyme can also catalyze the 2,3-bisphosphoglycerate to the 3-phosphoglycerate. In this study, the reaction mechanisms of both the phosphatase and the synthase activities of human bisphosphoglycerate mutase were theoretically calculated by using the quantum mechanics/molecular mechanics method based on the metadynamics and umbrella sampling simulations. The simulation results not only show the free energy curve of the phosphatase and the synthase reactions, but also reveal the important role of some residues in the active site. Additionally, the energy barriers of the two reactions indicate that the activity of the synthase in human bisphosphoglycerate mutase is much higher than that of the phosphatase. The estimated reaction barriers are consistent with the experimental data. Therefore, our work can give important information to understand the catalytic mechanism of the bisphosphoglycerate mutase family.

Constant pH molecular dynamics (CpHMD) and mutation studies: insights into AaegOBP1 pH-induced ligand releasing mechanism.

AaegOBP1, isolated from the male and female antenna of yellow fever mosquitoes, may serve as crucial molecular targets for the development of mosquitoes' attractants and for the control of mosquito populations. Nowadays crystal structures of AaegOBP1 in the neutral environment have been obtained, whereas little research is focused on the conformational change of AaegOBP1 in the acid solution. In our study, the conformational change and the ligand bound poses in different solution pH were investigated using constant pH molecular dynamics (CpHMD) as well as mutation studies. Results demonstrate that the protein changes dramatically in low pH solution and that the pH-sensing triad (Arg23-Tyr54-Ile125) plays an indispensable role in the structural stability and ligand binding. In addition, we have proved that the residue Arg23 is the most important one of the pH-sensing triad. This work could provide more penetrating understanding of the pH-induced ligand-releasing mechanism.

Exploring the molecular basis of dsRNA recognition by Mss116p using molecular dynamics simulations and free-energy calculations.

DEAD-box proteins are the largest family of helicase that are important in nearly all aspects of RNA metabolism. However, it is unclear how these proteins recognize and bind RNA. Here, we present a detailed analysis of the related DEAD-box protein Mss116p-RNA interaction, using molecular dynamics simulations with MM-GBSA calculations. The energetic analysis indicates that the two strands of double strands RNA (dsRNA) are recognized asymmetrically by Mss116p. The strand 1 of dsRNA provides the main binding affinity. Meanwhile, the nonpolar interaction provides the main driving force for the binding process. Although the contribution of polar interaction is small, it is vital in stabilizing the protein-RNA interaction. Compared with the wild type Mss116p, two studied mutants Q412A and D441A have obviously reduced binding free energies with dsRNA because of the decreasing of polar interaction. Three important residues Lys409, Arg415 and Arg438 lose their binding affinity significantly in mutants. In conclusion, these results complement previous experiments to advance comprehensive understanding of Mss116p-dsRNA interaction. The results also would provide support for the application of similar approaches to the understanding of other DEAD-box protein-RNA complexes.

Effects of Mass Change on Liquid-Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma.

In recent years, many experimental and theoretical studies of protein liquid-liquid phase separation (LLPS) have shown its important role in the processes of physiology and pathology. However, there is a lack of definite information on the regulation mechanism of LLPS in vital activities. Recently, we found that the intrinsically disordered proteins with the insertion/deletion of a non-interacting peptide segment or upon isotope replacement could form droplets, and the LLPS states are different from the proteins without those. We believed that there is an opportunity to decipher the LLPS mechanism with the mass change perspective. To investigate the effect of molecular mass on LLPS, we developed a coarse-grained model with different bead masses, including mass 1.0, mass 1.1, mass 1.2, mass 1.3, and mass 1.5 in atomic units or with the insertion of a non-interacting peptide (10 aa) and performed molecular dynamic simulations. Consequently, we found that the mass increase promotes the LLPS stability, which is based on decreasing the z motion rate and increasing the density and the inter-chain interaction of droplets. This insight into LLPS by mass change paves the way for the regulation and relevant diseases on LLPS.

Uncovering the Quantitative Relationships Among Chromosome Fluctuations, Epigenetics, and Gene Expressions of Transdifferentiation on Waddington Landscape.

The 3D spatial organization of the chromosomes appears to be linked to the gene function, which is cell type-specific. The chromosome structural ensemble switching model (CSESM) is developed by employing a heteropolymer model on different cell types and the important quantitative relationships among the chromosome ensemble, the epigenetic marks, and the gene expressions are uncovered, that both chromosome fluctuation and epigenetic marks have strong linear correlations with the gene expressions. The results support that the two compartments have different behaviors, corresponding to the relatively sparse and fluctuating phase (compartment A) and the relatively dense and stable phase (compartment B). Importantly, through the investigation of the transdifferentiation processes between the peripheral blood mononuclear cell (PBMC) and the bipolar neuron (BN), a quantitative description for the transdifferentiation is provided, which can be linked to the Waddington landscape. In addition, compared to the direct transdifferentiation between PBMC and BN, the transdifferentiation via the intermediate state neural progenitor cell (NPC) follows a different path (an "uphill" followed by a "downhill"). These theoretical studies bridge the gap among the chromosome fluctuations/ensembles, the epigenetics, and gene expressions in determining the cell fate.

Charge Interactions Modulate the Encounter Complex Ensemble of Two Differently Charged Disordered Protein Partners of KIX.

Disordered proteins play important roles in cell signaling and are frequently involved in protein-protein interactions. They also have a larger proportion of charged and polar residues than their folded counterparts. Here, we developed a structure-based model and applied molecular dynamics simulations to examine the presence and importance of electrostatic interactions in the binding processes of two differently charged intrinsically disordered ligands of the KIX domain of CBP. We observed non-native opposite-charged contacts in the encounter complexes for both ligands with KIX, and this may be a general feature of coupled folding and binding reactions. The ensemble of successful encounter complexes is a diverse set of structures, and in the case of the highly charged ligand, this ensemble was found to be malleable with respect to ionic strength. There are only minor differences between encounter complex ensembles for successful and unsuccessful collisions with no key interactions that appear to make the process far more productive. The energy landscape at this early stage in the process does not appear highly funneled. Strikingly we observed many native interactions that appear to reduce chances of an encounter complex being productive. Instead it appears that collectively non-native electrostatic interactions in the encounter complex increase the likelihood of productivity by holding the proteins together long enough for folding to take place. This mechanism is more effective for the more highly charged ligand.

Binding-Induced Conformational Changes Involved in Sliding Clamp PCNA and DNA Polymerase DPO4.

Cooperation between DNA polymerases and DNA sliding clamp proteins is essential for DNA replication and repair. However, it is still challenging to clarify the binding mechanism and the movements of Y-family DNA polymerase IV (DPO4) on the proliferating cell nuclear antigen (PCNA) ring. Here we develop the simulation models of DPO4-PCNA123 and DPO4-PCNA12 complexes and uncover the underlying dynamics of DPO4 during binding and the binding order of the DPO4 domains. Two important intermediate states are found on the free energy surface before reaching the final bound state. Our results suggest that both PCNA3 and DPO4 can influence the PCNA12 planar conformation, whereas the impact of PCNA3 on PCNA12 is more significant than DPO4. These findings provide the crucial information of the conformational dynamics of DPO4 and PCNA, as well as the clue of the underlying mechanism of the cooperation between DPO4 and PCNA during DNA replication.

Quantifying the Intrinsic Conformation Energy Landscape Topography of Proteins with Large-Scale Open-Closed Transition.

Large-scale conformational changes of proteins, including the open-closed transitions, are crucial for a variety of protein functions. These open-closed transitions are often associated with ligand binding. However, the understandings of the underlying mechanisms of the conformational changes within proteins during the open-closed transitions are still challenging at present. In this study, we quantified the intrinsic underlying conformational energy landscapes of five different proteins with large-scale open-closed transitions. This is realized by exploring the underlying density of states and the intrinsic conformational energy landscape topography measure Λ. Λ is a dimensionless ratio of conformational energy gap δE versus conformational energy roughness δE and configurational entropy S or size of the intrinsic conformational energy landscape. By quantifying the Λ of intrinsic open-closed conformational (Λoc) and intrinsic global folding (Λglobal) energy landscapes, we show that both intrinsic open-closed conformation energy and entropy landscapes are funneled toward the closed state. Furthermore, our results indicate the strong correlations between Λ and thermodynamics (conformational state transition temperature against trapping temperature) as well as between Λ and kinetics (open-closed kinetic time) of these proteins. This shows that the intrinsic conformational landscape topography determines both the conformational thermodynamic stability and kinetic speed of the conformational dynamics. Our investigations provide important insights for understanding the fundamental mechanisms of the protein conformational dynamics in a physical and global way.

Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes.

Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the "hot-spot" within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

Mutation and low pH effect on the stability as well as unfolding kinetics of transthyretin dimer.

Transthyretin (TTR) dissociation and aggregation appear to cause several amyloid diseases. TTR dimer is an important intermediate that is hard to be observed from the biological experiments. To date, the molecular origin and the structural motifs for TTR dimer dissociation, as well as the unfolding process have not been rationalized at atomic resolution. To this end, we have investigated the effect of low pH and mutation L55P on stability as well as the unfolding pathway of TTR dimer using constant pH molecular dynamics simulations. The result shows that acidic environment results in loose TTR dimer structure. Mutation L55P causes the disruption of strand D and makes the CE-loop very flexible. In acidic conditions, dimeric L55P mutant exhibits notable conformation changes and an evident trend to separate. Our work shows that the movements of strand C and the loops nearby are the beginning of the unfolding process. In addition, hydrogen bond network at the interface of the two monomers plays a part in stabilizing TTR dimer. The dynamic investigation on TTR dimer provides important insights into the structure-function relationships of TTR, and rationalizes the structural origin for the tendency of unfolding and changes of structure that occur upon introduction of mutation and pH along the TTR dimer dissociation and unfolding process.

Fosfomycin induced structural change in fosfomycin resistance kinases FomA: molecular dynamics and molecular docking studies.

Fosfomycin resistance kinases FomA, one of the key enzymes responsible for bacterial resistances to fosfomycin, has gained much attention recently due to the raising public concern for multi-drug resistant bacteria. Using molecular docking followed by molecular dynamics simulations, our group illustrated the process of fosfomycin induced conformational change of FomA. The detailed roles of the catalytic residues (Lys18, His58 and Thr210) during the formation of the enzyme-substrate complex were shown in our research. The organization functions of Gly53, Gly54, Ile61 and Leu75 were also highlighted. Furthermore, the cation-π interaction between Arg62 and Trp207 was observed and speculated to play an auxiliary role in the conformation change process of the enzyme. This detailed molecular level illustration of the formation of FomA·ATP·Mg·Fosfomycin complex could provide insight for both anti-biotic discovery and improvement of fosfomycin in the future.