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.

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.

Calculating 13 C NMR chemical shifts of large molecules using the eXtended ONIOM method at high accuracy with a low cost.

Fragmentation-based methods for nuclear magnetic resonance (NMR) chemical shift calculations have become more and more popular in first-principles calculations of large molecules. However, there are many options for a fragmentation-based method to select, such as theoretical methods, fragmentation schemes, the number of levels of theory, etc. It is important to study the optimal combination of the options to achieve a good balance between accuracy and efficiency. Here we investigate different combinations of options used by a fragmentation-based method, the eXtended ONIOM (XO) method, for 13 C chemical shift calculations on a set of organic and biological molecules. We found that: (1) introducing Hartree-Fock exchange into density functional theory (DFT) could reduce the calculation error due to fragmentation in contrast to pure DFT functionals, while a hybrid functional, xOPBE, is generally recommended; (2) fragmentation schemes generated from the molecular tailoring approach (MTA) with small level parameter n, for example, n = 2 and the degree-based fragmentation method (DBFM) with n = 1, are sufficient to achieve satisfactory accuracy; (3) the two-level XO (XO2) NMR calculation is superior to the calculation with only one level of theory, as the second level (i.e., low level) of theory provides a way to well describe the long-range effect. These findings are beneficial to practical applications of fragmentation-based methods for NMR chemical shift calculations of large molecules.

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.

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.

The enhanced extended phenomenological kinetics method to deal with timescale disparity problem among different reaction pathways.

Kinetic Monte Carlo method can provide valuable mechanistic insights for catalytic systems. Nonetheless, it suffers from the notorious problem of timescale disparity due to the existence of the complex catalytic network that consists of fast events and slow events. Previously, we have proposed the extended phenomenological kinetics (XPK) method that effectively deals with the timescale disparity problem between diffusion and reaction. However, it remains a great challenge to simulate systems with timescale disparity among different reaction pathways, which is important when selectivity is the major concern. In this study, we implement the enhanced XPK method to address this problem. The new algorithm works by identifying states connected through fast transitions and compressing them into a "superstate" when the chosen states satisfy a local steadystate condition. This state compression algorithm simplifies the reaction network by concealing the fast transitions. The accuracy and efficiency of the algorithm are demonstrated by two model systems: selective catalytic hydrogenation and selective catalytic decomposition. The enhanced XPK method is expected to be beneficial to the kinetic simulations of catalytic systems, especially those with complex reaction networks.

An unconventional ligand-binding mechanism of substrate-binding proteins: MD simulation and Markov state model analysis of BtuF.

In conventional "Venus Flytrap" mechanism, substrate-binding proteins (SBPs) interconvert between the open and closed conformations. Upon ligand binding, SBPs form a tightly closed conformation with the ligand bound at the interface of two domains. This mechanism was later challenged by many type III SBPs, such as the vitamin B12 -binding protein BtuF, in which the apo- and holo-state proteins adopt very similar conformations. Here, we combined molecular dynamics simulation and Markov state model analysis to study the conformational dynamics of apo- and B12 -bound BtuF. The results indicate that the crystal structures represent the only stable conformation of BtuF. Meanwhile, both apo- and holo-BtuF undergo large-scale interdomain motions with little energy cost. B12 binding casts little restraints on the interdomain motions, suggesting that ligand binding affinity is enhanced by the remaining conformational entropy of holo-BtuF. These results reveal a new paradigm of ligand recognition mechanism of SBPs. © 2019 Wiley Periodicals, Inc.

Structural Features and Energetics of the Periplasmic Entrance Opening of the Outer Membrane Channel TolC Revealed by Molecular Dynamics Simulation and Markov State Model Analysis.

TolC is a channel protein responsible for substrate translocation across the outer membrane, and it is also a part of the tripartite multidrug efflux pumps in Gram-negative bacteria. The crystal structure of TolC shows that the periplasmic entrance is tightly closed in the resting state, while substrate translocation definitely requires the entrance to open. How the occluded periplasmic entrance opens to allow passage of substrates remains elusive. In this work, we constructed a Markov state model from swarms of all-atom molecular dynamics (MD) simulation trajectories, which delineates the energetics of the conformational changes of TolC. Opening of the periplasmic entrance results in a monotonic increase in free energy and is accompanied by disruption of interprotomer interactions, whereas the intraprotomer interactions remain intact. Multi-ion potential of mean force (PMF) profiles for Na+ and Cl- permeation along the channel have been calculated, and the cation/anion permeability ratio derived from which are in good agreement with electrophysiological experiments. These results not only deepen our understanding of conformational dynamics of isolated TolC but also provide valuable vision of its functioning state in tripartite efflux pumps.

A Residue outside the Binding Site Determines the Gα Binding Specificity of GoLoco Motifs.

GoLoco motif-containing proteins regulate the nucleotide-binding state of Gα proteins in various signaling pathways. As guanine nucleotide dissociation inhibitors (GDIs), they bind Gα·GDP and inhibit GDP to GTP exchange. GoLoco proteins show binding selectivity toward different members of the Gα family. Although the Gαi1·GDP/RGS14 crystal structure explains the specific binding selectivity of the RGS14 GoLoco domain well, the mechanism of selective binding has not been understood for the more general features of short GoLoco domains found in tandem arrays in proteins like GPSM2/LGN/ dPins and GPSM1/AGS3. We explored the mechanism of differential interactions of GoLoco protein LGN with hGαi3 and hGαo. By combining mutagenesis experiments and molecular dynamics simulations, we identified a residue (Asp229 in hGαi3) away from the binding interface that remarkably affects the interaction between LGN and hGαi/o. A negatively charged residue at this position is required for high binding affinity. This affinity regulation mechanism was further verified by the cases of hGαi2 and dGαo, suggesting that this pathway is conserved among members of the Gα family.

A revisit of the conformational dynamics of SNARE protein rYkt6.

N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in the fusion of vesicles with their target membranes. R-SNARE protein Ykt6 is one of the most conserved SNARE in eukaryotes. The conformational state of Ykt6 is regulated by the lipidations at its C-terminal motif. Previous studies show that the binding of dodecylphosphocholine (DPC) can stabilize a closed conformation of rat Ykt6 (rYkt6) and mimic the farnesylated rYkt6. Despite this model, the detailed conformational dynamics of Ykt6 is still unclear. Here, we combined smFRET and MD simulation to demonstrate that the un-lipidated rYkt6 adopts five major conformational states. DPC binding shifts the conformational distribution toward the more closed states. At the same time, there remain considerable fractions of open and semi-open conformations in the presence of DPC. These newly revealed dynamic features of rYkt6 are consistent with its unique functional diversity in neuronal cells.

Lipid-Induced conformational switch controls fusion activity of longin domain SNARE Ykt6.

While most SNAREs are permanently anchored to membranes by their transmembrane domains, the dually lipidated SNARE Ykt6 is found both on intracellular membranes and in the cytosol. The cytosolic Ykt6 is inactive due to the autoinhibition of the SNARE core by its longin domain, although the molecular basis of this inhibition is unknown. Here, we demonstrate that unlipidated Ykt6 adopts multiple conformations, with a small population in the closed state. The structure of Ykt6 in complex with a fatty acid suggests that, upon farnesylation, the Ykt6 SNARE core forms four alpha helices that wrap around the longin domain, forming a dominantly closed conformation. The fatty acid, buried in a hydrophobic groove formed between the longin domain and its SNARE core, is essential for maintaining the autoinhibited conformation of Ykt6. Our study reveals that the posttranslationally attached farnesyl group can actively regulate Ykt6 fusion activity in addition to its anticipated membrane-anchoring role.

Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2.

Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called "pretranslocation" ("pre-T") state with a partially closed conformation suggest that the formation of this MalE-stabilized intermediate state is a key step leading to the outward-facing catalytic state. On the contrary, crosslinking and fluorescence studies suggest that ATP binding alone is sufficient to promote the outward-facing catalytic state, thereby doubting the role of MalE binding. To clarify the role of MalE binding and to gain deeper understanding of the molecular mechanisms of MalFGK2, we calculated the free energy surfaces (FESs) related to the lateral motion in the presence and absence of MalE using atomistic metadynamics simulations. The results showed that, in the absence of MalE, laterally closing motion was energetically forbidden but, upon MalE binding, more closed conformations similar to the pre-T state become more stable. The significant effect of MalE binding on the free energy landscapes was in agreement with crystallographic studies and confirmed the important role of MalE in stabilizing the pre-T state. Our simulations also revealed that the allosteric effect of MalE stimulation originates from the MalE-binding-promoted vertical motion between MalF and MalG cores, which was further supported by MD simulation of the MalE-independent mutant MalF500.

The Dynamic Multisite Interactions between Two Intrinsically Disordered Proteins.

Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well-characterized folding upon binding to dynamic fuzzy complexes. To date, most studies concern the binding of an IDP to a structured protein, while the interaction between two IDPs is poorly understood. In this study, NMR, smFRET, and molecular dynamics (MD) simulation are combined to characterize the interaction between two IDPs, the C-terminal domain (CTD) of protein 4.1G and the nuclear mitotic apparatus (NuMA) protein. It is revealed that CTD and NuMA form a fuzzy complex with remaining structural disorder. Multiple binding sites on both proteins were identified by molecular dynamics and mutagenesis studies. This study provides an atomic scenario in which two IDPs bearing multiple binding sites interact with each other in dynamic equilibrium. The combined approach employed here could be widely applicable for investigating IDPs and their dynamic interactions.

Conformational Dynamics and Protein-Substrate Interaction of ABC Transporter BtuCD at the Occluded State Revealed by Molecular Dynamics Simulations.

ATP-binding cassette (ABC) transporters are ubiquitous in all three kingdoms of life and are implicated in many clinically relevant physiological processes. They couple the energy released by ATP hydrolysis to facilitate substrate translocation across cell membranes. The crystal structures of type II ABC importers have revealed their unique transmembrane domain architecture consisting of 10 transmembrane helices and their structurally conserved nucleotide-binding domains among all ABC transporters. However, molecular details of the interactions between the importers and their substrate remain largely elusive. Taking vitamin B12 importer BtuCD as an exemplar of type II importers, we investigated the dynamics of its occluded state and the detailed protein-substrate interactions using molecular dynamics simulation. Our trajectories show that the importer accommodates the substrate through a nonspecific binding mode as the substrate undergoes evident vertical and tilt motions inside the translocation cavity. Extensive hydrogen bond and hydrophobic interactions were observed between the substrate and the importer; however, most of these interactions are weak, with <38% occurrence. The presence of substrate leads to enlargement of the translocation cavity, especially at its cytoplasmic end, which may activate cytoplasmic regions and probably facilitate the transportation. The perturbations caused by periplasmic binding protein and nucleotides were also investigated. The study provides deeper insight into the translocation mechanism of BtuCD.

The V-motifs facilitate the substrate capturing step of the PTS elevator mechanism.

We propose that the alternative crystal forms of outward open UlaA (which are experimental, not simulated, and contain the substrate in the cavity) can be used to interpret/validate the MD results from MalT (the substrate capture step, which involves the mobile second TMSs of the V-motifs, TMSs 2 and 7). Since the crystal contacts are the same between the two alternative crystal forms of outward open UlaA, the striking biological differences noted, including rearranged hydrogen bonds and salt bridge coordination, are not attributable to crystal packing differences. Using transport assays, we identified G58 and G286 as essential for normal vitamin C transport, but the comparison of alternative crystal forms revealed that these residues to unhinge TMS movements from substrate-binding side chains, rendering the mid-TMS regions of homologous TMSs 2 and 7 relatively immobile. While the TMS that is involved in substrate binding in MalT is part of the homologous bundle that holds the two separate halves of the transport assembly (two proteins) together, an unequal effect of the two knockouts was observed for UlaA where both V-motifs are free from such dimer interface interactions.

Lipid regulated conformational dynamics of the longin SNARE protein Ykt6 revealed by molecular dynamics simulations.

The conformation and subcellular localization of R-SNARE protein Ykt6 are regulated by the lipidation state of its C-terminal CCAIM motif. Biochemical and crystallography studies showed that lipid molecules binding at a hydrophobic pocket at the interface between the longin domain and the SNARE core can lock Ykt6 at a closed conformation and mimic the farnesylated state of Ykt6. In this study, we performed in silico farnesylation of Ykt6 and explored the conformational dynamics of Ykt6 using conventional and steered MD simulations. We found that the farnesylated Ykt6 model structure is stable during the 2 µs simulation and the farnesyl group adopts conformations similar to those of the DPC molecule bound to Ykt6. Both DPC binding and farnesylation were found to reduce the conformational flexibility of Ykt6 and hinder the dissociation of SNARE core from the longin domain. The dissociation of the αF-αG segment is the rate-limiting step during the putative closed-to-open conformational transition of Ykt6, and the key residues involved in this process are consistent with the experimental mutagenesis study.

Multiple conformational states and gate opening of outer membrane protein TolC revealed by molecular dynamics simulations.

Outer membrane protein TolC serves as an exit duct for exporting substances out of cell. The occluded periplasmic entrance of TolC is required to open for substrate transport, although the opening mechanism remains elusive. In this study, systematic molecular dynamics (MD) simulations for wild type TolC and six mutants were performed to explore the conformational dynamics of TolC. The periplasmic gate was shown to sample multiple conformational states with various degrees of gating opening. The gate opening was facilitated by all mutations except Y362F, which adopts an even more closed state than wild type TolC. The interprotomer salt-bridge R367-D153 is turned out to be crucial for periplasmic gate opening. The mutations that disrupt the interactions at the periplasmic tip may affect the stability of the trimeric assembly of TolC. Structural asymmetry of the periplasmic gate was observed to be opening size dependent. Asymmetric conformations are found in moderately opening states, while the most and the least opening states are often more symmetric. Finally, it is shown that lowering pH can remarkably stabilize the closed state of the periplasmic gate.

Molecular dynamics simulation of membrane proteins.

Membrane proteins play crucial roles in a range of biological processes. High resolution structures provide insights into the functional mechanisms of membrane proteins, but detailed biophysical characterization of membrane proteins is difficult. Complementary to experimental techniques, molecular dynamics simulations is a powerful tool in providing more complete description of the dynamics and energetics of membrane proteins with high spatial-temporal resolution. In this chapter, we provide a survey of the current methods and technique issues for setting up and running simulations of membrane proteins. The recent progress in applying simulations to understanding various biophysical properties of membrane proteins is outlined.

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.

The conformational transition pathway of ATP binding cassette transporter MsbA revealed by atomistic simulations.

ATP binding cassette transporters are integral membrane proteins that use the energy released from ATP hydrolysis at the two nucleotide binding domains (NBDs) to translocate a wide variety of substrates through a channel at the two transmembrane domains (TMDs) across the cell membranes. MsbA from Gram-negative bacteria is a lipid and multidrug resistance ATP binding cassette exporter that can undergo large scale conformational changes between the outward-facing and the inward-facing conformations revealed by crystal structures in different states. Here, we use targeted molecular dynamics simulation methods to explore the atomic details of the conformational transition from the outward-facing to the inward-facing states of MsbA. The molecular dynamics trajectories revealed a clear spatiotemporal order of the conformational movements. The disruption of the nucleotide binding sites at the NBD dimer interface is the very first event that initiates the following conformational changes, verifying the assumption that the conformational conversion is triggered by ATP hydrolysis. The conserved x-loops of the NBDs were identified to participate in the interaction network that stabilizes the cytoplasmic tetrahelix bundle of the TMDs and play an important role in mediating the cross-talk between the NBD and TMD. The movement of the NBD dimer is transmitted through x-loops to break the tetrahelix bundle, inducing the packing rearrangements of the transmembrane helices at the cytoplasmic side and the periplasmic side sequentially. The packing rearrangement within each periplasmic wing of TMD that results in exposure of the substrate binding sites occurred at the end stage of the trajectory, preventing the wrong timing of the binding site accessibility.