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Transmembrane protein 16F (TMEM16F) is a ubiquitously expressed Ca

Molecular motors have evolved to transduce chemical energy from adenosine triphosphate into mechanical work to drive essential cellular processes, from muscle contraction to vesicular transport. Dysfunction of these motors is a root cause of many pathologies necessitating the need for intrinsic control over molecular motor function. Herein, we demonstrate that positional isomerism can be used as a simple and powerful tool to control the molecular motor of muscle, myosin. Using three isomers of a synthetic non-nucleoside triphosphate we demonstrate that myosin’s force and motion generating capacity can be dramatically altered at both the ensemble and single molecule levels. By correlating our experimental results with computation, we show that each isomer exerts intrinsic control by affecting distinct steps in myosin’s mechano-chemical cycle. Our studies demonstrate that subtle variations in the structure of an abiotic energy source can be used to control the force and motility of myosin without altering myosin’s structure.Statement of SignificanceMolecular motors transduce chemical energy from ATP into the mechanical work inside a cell, powering everything from muscle contraction to vesicular transport. While ATP is the preferred source of energy, there is growing interest in developing alternative sources of energy to gain control over molecular motors. We synthesized a series of synthetic compounds to serve as alternative energy sources for muscle myosin. Myosin was able to use this energy source to generate force and velocity. And by using different isomers of this compound we were able to modulate, and even inhibit, the activity of myosin. This suggests that changing the isomer of the substrate could provide a simple, yet powerful, approach to gain control over molecular motor function.

Mitochondrial permeability transition pore (mPTP) plays crucial roles in cell death in a variety of diseases, including ischemia/reperfusion injury in heart attack and stroke, neurodegenerative conditions, and cancer. To date, cyclophilin D is the only confirmed component of mPTP. Under stress, p53 can translocate into mitochondria and interact with CypD, triggering necrosis and cell growth arrest. However, the molecular details of p53/CypD interaction are still poorly understood. Previously, several studies reported that p53 interacts with CypD through its DNA-binding domain (DBD). However, using surface plasmon resonance (SPR), we found that full-length p53 (FLp53) binds to CypD with KD of ~1 μM, while both NTD-DBD and NTD bind to CypD at ~10 μM KD (Fig. 1C and 1D). Thus, instead of DBD, NTD is the major CypD binding site on p53. NMR titration and MD simulation revealed that NTD binds CypD with broad and dynamic interfaces dominated by electrostatic interactions. NTD 20-70 was further identified as the minimal binding region for CypD interaction, and two NTD fragments, D1 (residues 22-44) and D2 (58-70), can each bind CypD with mM affinity. Our detailed biophysical characterization of the dynamic interface between NTD and CypD provides novel insights on the p53-dependent mPTP opening and drug discovery targeting NTD/CypD interface in diseases.

Aggregation of amyloid-β (Aβ) peptides from soluble monomers to insoluble amyloid fibrils has been hypothesized to be one of the crucial steps in the progression of Alzheimer’s disease (AD). Due to the disordered nature of Aβ peptides, identifying aggregation inhibitors as potential drug candidates against AD has been a great challenge. In this communication, we report an atomistic simulation study of the inhibition mechanism of two small molecules, homotaurine and scyllo-inositol, which are AD drug candidates currently under investigation. Using a replica exchange molecular dynamics method to extensively explore the conformational space of Aβ42 monomer with and without the presence of the small molecule agents, we found that both drug candidates reduce the β-strand propensity of the C-terminus region (I31-A42) and promote a conformational change of Aβ42 monomer toward a more collapsed phase through a non-specific binding mechanism. These findings provide atomistic-level insights into understanding of the inhibitory mechanisms of the two potential small-molecule drug candidates for AD treatment in the future.

The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability, smooth muscle contraction, fluid secretion and gut motility. While opening of TMEM16A requires binding of intracellular Ca2+, prolonged Ca2+-dependent activation results in channel desensitization or rundown, the mechanism of which is unclear. Here we show that phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization via binding to a putative binding site at the cytosolic interface of transmembrane segments (TMs) 3–5. We further demonstrate that the ion-conducting pore of TMEM16A is constituted of two functionally distinct modules: a Ca2+-binding module formed by TMs 6–8 and a PIP2-binding regulatory module formed by TMs 3–5, which mediate channel activation and desensitization, respectively. PIP2 dissociation from the regulatory module results in ion-conducting pore collapse and subsequent channel desensitization. Our findings thus provide key insights into the mechanistic understanding of TMEM16 channel gating and lipid-dependent regulation., The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability and muscle contraction. Here authors show that phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization via binding to a putative binding site.

Intrinsically disordered proteins (IDPs) often fold into stable structures upon specific binding. The roles of residual structure of unbound IDPs in coupling binding and folding have been under much debate. While many studies emphasize the importance of conformational flexibility for IDP recognition, it was recently demonstrated that stabilization the N-terminal helix of intrinsically disordered ACTR accelerated its binding to another IDP, NCBD of the CREB-binding protein. To understand how enhancing ACTR helicity accelerates binding, we derived a series of topology-based coarse-grained models that mimicked various ACTR mutants with increasing helical contents and reproduced their NCBD binding affinities. Molecular dynamics simulations were then performed to sample hundreds of reversible coupled binding and folding transitions. The results show that increasing ACTR helicity does not alter the baseline mechanism of synergistic folding, which continues to follow “extended conformational selection” with multiple stages of selection and induced folding. Importantly, these coarse-grained models, while only calibrated based on binding thermodynamics, recapitulate the observed kinetic acceleration with increasing ACTR helicity. However, the residual helices do not enhance the association kinetics via more efficient seeding of productive collisions. Instead, they allow the nonspecific collision complexes to evolve more efficiently into the final bound and folded state, which is the primary source of accelerated association kinetics. Meanwhile, reduced dissociation kinetics with increasing ACTR helicity can be directly attributed to smaller entropic cost of forming the bound state. Altogether, this study provides important mechanistic insights into how residual structure may modulate thermodynamics and kinetics of IDP interactions.

Epigallocatechin gallate (EGCG) from green tea can induce apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Using SPR and NMR, here we report a direct, μM interaction between EGCG and the tumor suppressor p53 (KD = 1.6 ± 1.4 μM), with the disordered N-terminal domain (NTD) identified as the major binding site (KD = 4 ± 2 μM). Large scale atomistic simulations (>100 μs), SAXS and AUC demonstrate that EGCG-NTD interaction is dynamic and EGCG causes the emergence of a subpopulation of compact bound conformations. The EGCG-p53 interaction disrupts p53 interaction with its regulatory E3 ligase MDM2 and inhibits ubiquitination of p53 by MDM2 in an in vitro ubiquitination assay, likely stabilizing p53 for anti-tumor activity. Our work provides insights into the mechanisms for EGCG’s anticancer activity and identifies p53 NTD as a target for cancer drug discovery through dynamic interactions with small molecules., Epigallocatechin gallate (EGCG) is a catechin flavonoid which induces apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Here authors use an interdisciplinary approach to show a direct interaction between EGCG and the tumor suppressor p53 and demonstrate that EGCG inhibits ubiquitination of p53 by MDM2.

Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue ‘C-linker’. To determine the linker’s role in human BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combining atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.

TMEM16A is a widely expressed Ca2+-activated Cl− channel that regulates crucial physiological functions including fluid secretion, neuronal excitability, and smooth muscle contraction. There is a critical need to understand the molecular mechanisms of TMEM16A gating and regulation. However, high-resolution TMEM16A structures have failed to reveal an activated state with an unobstructed permeation pathway even with saturating Ca2+. This has been attributed to the requirement of PIP2 for preventing TMEM16A desensitization. Here, atomistic simulations show that specific binding of PIP2 to TMEM16A can lead to spontaneous opening of the permeation pathway in the Ca2+-bound state. The predicted activated state is highly consistent with a wide range of mutagenesis and functional data. It yields a maximal Cl− conductance of ~1 pS, similar to experimental estimates, and recapitulates the selectivity of larger SCN− over Cl−. The resulting molecular mechanism of activation provides a basis for understanding the interplay of multiple signals in controlling TMEM16A channel function. Chen and Jia investigate the synergistic regulating role of Ca2+ binding and the signaling lipid PIP2 in TMEM16A channel gating. Their study is significant as it provides new insights into the activated state of TMEM16A and highlights an example of functional importance of lipids in regulating membrane-associated proteins.

Conformational changes of proteins are essential to their functions. Yet it remains challenging to measure the amplitudes and timescales of protein motions. Here we show that the cytolysin A (ClyA) nanopore was used as a molecular tweezer to trap a single maltose-binding protein (MBP) within its lumen, which allows conformation changes to be monitored as electrical current fluctuations in real time. In contrast to the current two state binding model, the current measurements revealed three distinct ligand-bound states for MBP in the presence of reducing saccharides. Our analysis reveal that these three states represented MBP bound to different isomers of reducing sugars. These findings contribute to on the understanding of the mechanism of substrate recognition by MBP and illustrate that the nanopore tweezer is a powerful, label-free, single-molecule approach for studying protein conformational dynamics under functional conditions.

Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue "C-linker". To determine the linker's role in BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combing atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results also suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.

Anti-apoptotic Bcl-xL plays central roles in regulating programed cell death. Partial unfolding of Bcl-xL has been observed at the interface upon specific binding to the pro-apoptotic BH3-only protein PUMA, which in turn disrupts the interaction of Bcl-xL with tumor suppressor p53 and promotes apoptosis. Previous analysis of existing Bcl-xL structures and atomistic molecular dynamics (MD) simulations have suggested that substantial intrinsic structure heterogeneity exists at the BH3-only protein binding interface of Bcl-xL to facilitate its conformational transitions upon binding. In this study, enhanced sampling is applied to further characterize the interfacial conformations of unbound Bcl-xL in explicit solvent. Extensive replica exchange with solute tempering (REST) simulations, with a total accumulated time of 16 μs, were able to cover much wider conformational spaces for the interfacial region of Bcl-xL. The resulting structural ensembles are much better converged, with local and long-range structural features that are highly consistent with existing NMR data. These simulations further demonstrate that the BH3-only protein binding interface of Bcl-xL is intrinsically disordered and samples many rapidly interconverting conformations. Intriguingly, all previously observed conformers are well represented in the unbound structure ensemble. Such intrinsic structural heterogeneity and flexibility may be critical for Bcl-xL to undergo partial unfolding induced by PUMA binding, and likely provide a robust basis that allows Bcl-xL to respond sensitively to binding of various ligands in cellular signaling and regulation.

Intrinsically disordered proteins (IDPs) are important functional proteins, and their deregulation are linked to numerous human diseases including cancers. Understanding how disease-associated mutations or drug molecules can perturb the sequence-disordered ensemble-function-disease relationship of IDPs remains challenging, because it requires detailed characterization of the heterogeneous structural ensembles of IDPs. In this work, we combine the latest atomistic force field a99SB-disp, enhanced sampling technique replica exchange with solute tempering, and GPU-accelerated molecular dynamics simulations to investigate how four cancer-associated mutations, K24N, N29K/N30D, D49Y, and W53G, and binding of an anti-cancer molecule, epigallocatechin gallate (EGCG), modulate the disordered ensemble of the transactivation domain (TAD) of tumor suppressor p53. Through extensive sampling, in excess of 1.0 μs per replica, well-converged structural ensembles of wild-type and mutant p53-TAD as well as WT p53-TAD in the presence of EGCG were generated. The results reveal that mutants could induce local structural changes and affect secondary structural properties. Interestingly, both EGCG binding and N29K/N30D could also induce long-range structural reorganizations and lead to more compact structures that could shield key binding sites of p53-TAD regulators. Further analysis reveals that the effects of EGCG binding are mainly achieved through nonspecific interactions. These observations are generally consistent with on-going NMR studies and binding assays. Our studies suggest that induced conformational collapse of IDPs may be a general mechanism for shielding functional sites, thus inhibiting recognition of their targets. The current study also demonstrates that atomistic simulations provide a viable approach for studying the sequence-disordered ensemble-function-disease relationships of IDPs and developing new drug design strategies targeting regulatory IDPs.

Conformational changes of proteins are essential to their functions. Yet it remains challenging to measure the amplitudes and timescales of protein motions. Here we show that the ClyA nanopore can be used as a molecular tweezer to trap a single maltose-binding protein (MBP) within its lumen, which allows conformation changes to be monitored as electrical current fluctuations in real time. The current measurements revealed three distinct ligand-bound states for MBP in the presence of reducing saccharides. Our biochemical and kinetic analysis reveal that these three states represented MBP bound to different isomers of reducing sugars. These findings shed light on the mechanism of substrate recognition by MBP and illustrate that the nanopore tweezer is a powerful, label-free, single-molecule approach for studying protein conformational dynamics under functional conditions.

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

Transmembrane protein 16F (TMEM16F) is an enigmatic Ca2+-activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. Despite recent advances in the structure and function understanding of TMEM16 proteins, how mammalian TMEM16 CaPLSases open and close, or gate their phospholipid permeation pathways remains unclear. Here we identify an inner activation gate, which is established by three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway of TMEM16F-CaPLSase. Disrupting the inner gate profoundly alters TMEM16F phospholipid permeation. Lysine substitutions of F518 and Y563 even lead to constitutively active CaPLSases that bypass Ca2+-dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca2+-activated Cl− channel (CaCC). The identification of an inner activation gate can help elucidate the gating and permeation mechanism of TMEM16 CaPLSases and channels., TMEM16F is an enigmatic Ca2 + -activated phospholipid scramblase (CaPLSase) that passively transports phospholipids. Here authors identify an inner activation gate and its disruption profoundly alters TMEM16F phospholipid permeation.

The gating mechanism of transmembrane ion channels is crucial for understanding how these proteins control ion flow across membranes in various physiological processes. Big potassium (BK) channels are particularly interesting with large single-channel conductance and dual regulation by membrane voltage and intracellular Ca2+. Recent atomistic structures of BK channels failed to identify structural features that could physically block the ion flow in the closed state. Here, we show that gating of BK channels does not seem to require a physical gate. Instead, changes in the pore shape and surface hydrophobicity in the Ca2+-free state allow the channel to readily undergo hydrophobic dewetting transitions, giving rise to a large free energy barrier for K+ permeation. Importantly, the dry pore remains physically open and is readily accessible to quaternary ammonium channel blockers. The hydrophobic gating mechanism is also consistent with scanning mutagenesis studies showing that modulation of pore hydrophobicity is correlated with activation properties., BK channels are regulated by membrane voltage and intracellular Ca2+ but the structural features that block the ion flow in the closed state remain unknown. Here authors use molecular dynamics simulation and show that a physical gate is not required; instead ion flow is regulated by hydrophobic dewetting due to changes in pore shape and surface hydrophobicity.

Calcium-activated chloride channels (CaCCs) are critical in regulating neural excitability, nociception, smooth muscle contraction, secretion and gut motility. Besides Ca2 - and voltage-dependent activation, another hallmark of TMEM16A-CaCC is time-dependent desensitization or rundown, the mechanism of which is unclear. Here we report that phosphatidylinositol-(4,5)bisphosphate (PIP2) controls TMEM16A desensitization by stabilizing the channel pore, which is formed by a PIP2 binding ‘regulatory module’ of transmembrane segments (TMs) 3-5 and a ‘Ca2 -binding module’ of TMs6-8. Under sub-micromolar Ca2 , PIP2 dissociation from the ‘regulatory module’ leads to transient pore collapse and desensitization, which can be rapidly reversed by exogenous PIP2, higher Ca2 or voltage. Sustained channel opening under saturating Ca2 requires PIP2 to prevent the persistent pore collapse and desensitization, whose recovery needs prolonged exogenous PIP2 exposure. The PIP2-dependent, bimodal desensitization mechanism and the proposed modular design of TMEM16A pore can shine lights on understanding the structure, function, regulation and physiology of TMEM16 family.

The ability of a transmembrane helix (TMH) to insert into a lipid bilayer has been mainly understood based on the total hydrophobicity of the peptide sequence. Recently, Hedin et al. investigated the influence of flanking loops on membrane insertion of a set of marginally hydrophobic TMHs using translocon-based membrane integration assays. While the flanking loops were found to facilitate the insertion in most cases, counter examples also emerged where the flanking loops hinder membrane insertion and contradict the hydrophobicity and charge distribution analyses. Here, coarse-grained free energy calculations and atomistic simulations were performed to investigate the energetics and conformational details of the membrane insertion of two representative marginally hydrophobic TMHs with (NhaL and EmrL) and without (NhaA and EmrD) the flanking loops. The simulations fail to directly recapitulate the contrasting effects of the flanking loops for these two TMHs, due to systematic over-prediction of the stabilities of the transmembrane states that has also been consistently observed in previous studies. Nonetheless, detailed force decomposition and peptide conformation analyses suggest a novel mechanism on how the peptide conformational equilibrium in the aqueous phase may modulate the effects of flanking loops on membrane insertion. Specifically, the flanking loops in peptide EmrL interact strongly with the TMH segment and form stable compact conformations in the aqueous phase, which can hinder membrane absorption and insertion as these processes require extended conformations with minimal interactions between the flanking loops and TMH segment. This work also emphasizes the general importance of considering the peptide conformational equilibrium for understanding the mechanism and energetics of membrane insertion, an aspect that has not yet been sufficiently addressed in the literature.

Intrinsically disordered proteins (IDPs) are now recognized to be prevalent in biology, and many potential functional benefits have been discussed. However, the frequent requirement of peptide folding in specific interactions of IDPs could impose a kinetic bottleneck, which could be overcome only by efficient folding upon encounter. Intriguingly, existing kinetic data suggest that specific binding of IDPs is generally no slower than that of globular proteins. Here, we exploited the cell cycle regulator p27(Kip1) (p27) as a model system to understand how IDPs might achieve efficient folding upon encounter for facile recognition. Combining experiments and coarse-grained modeling, we demonstrate that long-range electrostatic interactions between enriched charges on p27 and near its binding site on cyclin A not only enhance the encounter rate (i.e., electrostatic steering) but also promote folding-competent topologies in the encounter complexes, allowing rapid subsequent formation of short-range native interactions en route to the specific complex. In contrast, nonspecific hydrophobic interactions, while hardly affecting the encounter rate, can significantly reduce the efficiency of folding upon encounter and lead to slower binding kinetics. Further analysis of charge distributions in a set of known IDP complexes reveals that, although IDP binding sites tend to be more hydrophobic compared to the rest of the target surface, their vicinities are frequently enriched with charges to complement those on IDPs. This observation suggests that electrostatically accelerated encounter and induced folding might represent a prevalent mechanism for promoting facile IDP recognition.

The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK4-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity. This article is part of a Special Issue entitled: Protein Folding in Membranes.

We have developed a set of orientational restraint potentials for solid-state NMR observables including 15 N chemical shift and 15 N– 1 H dipolar coupling. Torsion angle molecular dynamics simulations with available experimental 15 N chemical shift and 15 N– 1 H dipolar coupling as target values have been performed to determine orientational information of four membrane proteins and to model the structures of some of these systems in oligomer states. The results suggest that incorporation of the orientational restraint potentials into molecular dynamics provides an efficient means to the determination of structures that optimally satisfy the experimental observables without an extensive geometrical search.

Peptide-based biomaterials have many potential applications in tissue engineering, drug delivery, surface engineering, and other areas. In this study, we exploited a series of amphiphilic diblock model peptides (L5K10, L5GSIIK10, and L5P(D)PK10) to understand how the supramolecular assembly morphology may be modulated by the physical properties of the peptide monomer and experimental conditions. A combination of experimentation and simulation revealed that although all three peptides lack stable structures as monomers, their levels of conformational heterogeneity differ significantly. Importantly, such differences appear to be correlated with the peptides' ability to form sheet-like assemblies. In particular, substantial conformational heterogeneity appears to be required for anisotropic growth of sheet-like materials, likely by reducing the peptide assembly kinetics. To test this hypothesis, we increased the pH to neutralize the lysine residues and promote peptide aggregation, and the resulting faster assembly rate hindered the growth of the sheet morphology as predicted. In addition, we designed and investigated the assembly morphologies of a series of diblock peptides with various lengths of polyglycine inserts, L5GxK10, x = 1, 2, 3, 4. The results further supported the importance of peptide conformational flexibility and pH in modulation of the peptide supramolecular assembly morphology.

Modeling pH-coupled conformational dynamics allows one to probe many important pH-dependent biological processes, ranging from ATP synthesis, enzyme catalysis, and membrane fusion to protein folding/misfolding and amyloid formation. This work illustrates the strengths and capabilities of continuous constant pH molecular dynamics in exploring pH-dependent conformational transitions in proteins by revisiting an experimentally well studied model protein fragment, the C peptide from ribonuclease A. The simulation data reveal a bell-shaped pH profile for the total helix content, in agreement with experiment, and several pairs of electrostatic interactions that control the relative populations of unfolded and partially folded states of various helical lengths. The latter information greatly complements and extends that attainable by current experimental techniques. The present work paves the way for new and exciting applications, such as the study of pH-dependent molecular mechanism in the formation of amyloid comprising peptides from Alzheimer's and Parkinson's diseases.

Successful protein folding in physiological timescales requires a biased free energy landscape to restrict the otherwise prohibitive search space. The existence of this bias is a necessary outcome of evolution since sequences that fold slowly do not contribute to the fitness of the organism. In contrast, the evolutionary pressure for efficient folding pathways is not present in the formation of pathological aggregates. Accordingly, the measured growth rates for amyloid fibrils are much slower than might be expected for the formation of beta sheets. Analytic theory shows that fibril growth rates are consistent with a random search over the alignments of intermolecular H-bonds and that solution conditions that accelerate this search (i.e. weakening bonds) can increase aggregation rates. This theory identifies two reaction coordinates, the alignment between molecules and the number of formed H-bonds, which we use to devise a novel Markov State Model to simulate fibril growth in atomistic detail. This model is used to simulate the growth of beta amyloid (16-22) and three mutants. The simulations qualitatively capture the non-additive effects of the mutations, but interestingly there is no obvious trend to the mutation effects in the lifetimes of the molecular alignments or individual H-bonds. Instead, the changes in the growth rate emerge from the accumulation of many small perturbations over a large ensemble of trajectories. We conclude with a discussion of theory development with an eye towards the simulation of very slow processes like fibril nucleation.

Significant progresses have been made in the design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the second transmembrane domain of the α-subunit of the glycine receptor (GlyR). The latest designs, including p22 (KKKKP ARVGL GITTV LTMTT QS), are highly soluble in water with minimal aggregation propensity and insert efficiently into cell membranes to form highly conductive ion channels. The last obstacle to a potential lead sequence for channel replacement treatment of CF patients is achieving adequate chloride selectivity. We have performed free energy simulation to analyze the conductance and charge selectivity of M2GlyR-derived synthetic channels. The results reveal that the pentameric p22 pore is non-selective. Moderate barriers for permeation of both K+ and Cl− are dominated by the desolvation cost. Despite previous evidence suggesting a potential role of threonine side chains in anion selectivity, the hydroxyl group is not a good surrogate of water for coordinating these ions. We have also tested initial ideas of introducing additional rings of positive changes to various positions along the pore to increase anion selectivity. The results support the feasibility of achieving anion selectivity by modifying the electrostatic properties of the pore, but at the same time suggest that the peptide assembly and pore topology may also be dramatically modified, which could abolish the effects of modified electrostatics on anion selectivity. This was confirmed by subsequent two-electrode voltage clamp measurements showing that none of the tested mono-, di- and tri-Dap substituted sequences was selective. The current study thus highlights the importance of controlling channel topology besides modifying pore electrostatics for achieving anion selectivity. Several strategies are now being explored in our continued efforts to design an anion selective peptide channel with suitable biophysical, physiological and pharmacological properties as a potential treatment modality for channel replacement therapy. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Intrinsically disordered proteins (IDPs) are a fascinating class of newly recognized proteins that can exist as dynamic and heterogeneous ensembles of disordered structures under physiological conditions (1). They are highly prevalent in biology, frequently play crucial roles in cell signaling and regulation, and are associated with numerous human diseases (2). Many concepts have been proposed on how intrinsic conformational disorder may offer functional advantages, such as structural plasticity for binding multiple partners and inducibility by posttranslational modifications (3).

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation., Author Summary Intrinsically disordered proteins (IDPs) are key components of regulatory networks that dictate various aspects of cellular decision-making. They are over-represented in major disease pathways, and are considered novel albeit currently difficult drug targets. Recognition of IDPs has extended the traditional protein structure-function paradigm, and various concepts have been proposed on how intrinsic disorder may confer crucial functional advantages. However, the physical basis of these concepts remains poorly established. In particular, while IDPs alone exist as ensembles of fluctuating structures, they frequently fold upon specific binding. Analysis of the physical timescales of protein folding and protein-protein encounter predicts that the requirement of peptide folding for specific binding could lead to a major kinetic bottleneck. In this work, carefully calibrated topology-based coarse-grained models were applied to directly simulate reversible folding and binding and investigate the recognition mechanisms of three IDP complexes. The results strongly support an electrostatically accelerated encounter and folding mechanism, where long-range electrostatic forces not only accelerate protein-protein encounter via “electrostatic steering” but also promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter.

Intrinsically disordered proteins (IDPs) are an important class of functional proteins that is highly prevalent in biology and has broad association with human diseases. In contrast to structured proteins, free IDPs exist as heterogeneous and dynamical conformational ensembles under physiological conditions. Many concepts have been discussed on how such intrinsic disorder may provide crucial functional advantages, particularly in cellular signaling and regulation. Establishing the physical basis of these proposed phenomena requires not only detailed characterization of the disordered conformational ensembles, but also mechanistic understanding of the roles of various ensemble properties in IDP interaction and regulation. Here, we review the experimental and computational approaches that may be integrated to address many important challenges of establishing a “structural” basis of IDP function, and discuss some of the key emerging ideas on how the conformational ensembles of IDPs may mediate function, especially in coupled binding and folding interactions.

Peptide-based packaging systems show great potential as safer drug delivery systems. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. Here, we describe a set of 15 & 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. These peptides undergo supramolecular self-assembly and form solvent-filled, bilayer delimited spheres with 50–200 nm diameters as confirmed by TEM, STEM and DLS. Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these all-peptide structures are stabilized potentially by both hydrophobic interactions and hydrogen bonds and remain intact at low micromolar concentrations and higher temperatures. A linear peptide lacking the branch point showed no self-assembly properties. We have observed that these peptide vesicles can trap fluorescent dye molecules within their interior and are taken up by N/N 1003A rabbit lens epithelial cells grown in culture. These assemblies are thus potential drug delivery systems that can overcome some of the key limitations of the current packaging systems.

To understand the interplay of residual structures and conformational fluctuations in the interaction of intrinsically disordered proteins (IDPs), we first combined implicit solvent and replica exchange sampling to calculate atomistic disordered ensembles of the nuclear co-activator binding domain (NCBD) of transcription coactivator CBP and the activation domain of the p160 steroid receptor coactivator ACTR. The calculated ensembles are in quantitative agreement with NMR-derived residue helicity and recapitulate the experimental observation that, while free ACTR largely lacks residual secondary structures, free NCBD is a molten globule with a helical content similar to that in the folded complex. Detailed conformational analysis reveals that free NCBD has an inherent ability to substantially sample all the helix configurations that have been previously observed either unbound or in complexes. Intriguingly, further high-temperature unbinding and unfolding simulations in implicit and explicit solvents emphasize the importance of conformational fluctuations in synergistic folding of NCBD with ACTR. A balance between preformed elements and conformational fluctuations appears necessary to allow NCBD to interact with different targets and fold into alternative conformations. Together with previous topology-based modeling and existing experimental data, the current simulations strongly support an “extended conformational selection” synergistic folding mechanism that involves a key intermediate state stabilized by interaction between the C-terminal helices of NCBD and ACTR. In addition, the atomistic simulations reveal the role of long-range as well as short-range electrostatic interactions in cooperating with readily fluctuating residual structures, which might enhance the encounter rate and promote efficient folding upon encounter for facile binding and folding interactions of IDPs. Thus, the current study not only provides a consistent mechanistic understanding of the NCBD/ACTR interaction, but also helps establish a multi-scale molecular modeling framework for understanding the structure, interaction, and regulation of IDPs in general., Author Summary Intrinsically disordered proteins (IDPs) are now widely recognized to play fundamental roles in biology and to be frequently associated with human diseases. Although the potential advantages of intrinsic disorder in cellular signaling and regulation have been widely discussed, the physical basis for these proposed phenomena remains sketchy at best. An integration of multi-scale molecular modeling and experimental characterization is necessary to uncover the molecular principles that govern the structure, interaction, and regulation of IDPs. In this work, we characterize the conformational properties of two IDPs involved in transcription regulation at the atomistic level and further examine the roles of these properties in their coupled binding and folding interactions. Our simulations suggest interplay among residual structures, conformational fluctuations, and electrostatic interactions that allows efficient synergistic folding of these two IDPs. In particular, we propose that electrostatic interactions might play an important role in facilitating rapid folding and binding recognition of IDPs, by enhancing the encounter rate and promoting efficient folding upon encounter.

Notch receptors are core components of the Notch signaling pathway and play a central role in cell fate decisions during development as well as tissue homeostasis. Upon ligand binding, Notch is sequentially cleaved at the S2 site by an ADAM protease and at the S3 site by the γ-secretase complex. Recent X-ray structures of the negative regulatory region (NRR) of the Notch receptor reveal an auto-inhibited fold where three protective Lin12/Notch repeats (LNR) of the NRR shield the S2 cleavage site housed in the heterodimerization (HD) domain. One of the models explaining how ligand binding drives the NRR conformation from a protease-resistant state to a protease-sensitive one invokes a mechanical force exerted on the NRR upon ligand endocytosis. Here, we combined physics-based atomistic simulations and topology-based coarse-grained modeling to investigate the intrinsic and force-induced folding and unfolding mechanisms of the human Notch1 NRR. The simulations support that external force applied to the termini of the NRR disengages the LNR modules from the heterodimerization (HD) domain in a well-defined, largely sequential manner. Importantly, the mechanical force can further drive local unfolding of the HD domain in a functionally relevant fashion that would provide full proteolytic access to the S2 site prior to heterodimer disassociation. We further analyzed local structural features, intrinsic folding free energy surfaces, and correlated motions of the HD domain. The results are consistent with a model in which the HD domain possesses inherent mechanosensing characteristics that could be utilized during Notch activation. This potential role of the HD domain in ligand-dependent Notch activation may have implications for understanding normal and aberrant Notch signaling.

The nuclear coactivator-binding domain (NCBD) of CREB binding protein is an intrinsically disordered protein (IDP), which exists as molten globule like structures in the unbound state. As one of the most folded IDPs, NCBD folds synergistically with another IDP, activator for thyroid hormone and retinoid receptor (ACTR) of the p160 steroid receptor. A topology-based Go-like coarse-grained protein model has been used to investigate the mechanism of the NCBD:ACTR interaction. The simulation results support a largely cooperative mechanism for the folding of the two IDPs. Specifically, while the binding induced folding follows multiple pathways, the α2 helix of ACTR most frequently initiates the binding by interacting with a preformed structural motif of NCBD, where the α2 and α3 helices of NCBD are mostly folded and correctly packed. This initial binding is followed by the binding and folding of rest of the helices of both IDPs in a highly cooperative fashion. This is consistent with the importance of the disordered leucine-rich motifs in specificity of NCBD:ACTR established by previous biochemical and biophysical data, and further supported by unpublished mass spectroscopy data from David Weis's lab. Compared to ACTR, folding of NCBD appear to mostly involve assembly of pre-folded of α1 and α2 helices, and only α3 folding appears to be initiated by ACTR binding.

Replica exchange molecular dynamics (REX-MD) is a generalized ensemble method, which periodically exchanges replicas between neighbor temperature windows to help cross energy barriers in energy space, therefore enhancing the sampling efficiency. It has been shown to be very effective on simple two-state model systems. However, for more complicated processes such as protein folding, REMD has not been adequately tested. In particular, in simulations of proteins in the current physics-based force fields, different replicas often end up trapped in segregated regions of the phase space, which significantly reduces the sampling efficiency. Here we systematically investigate the efficiency of REX sampling using simplified, yet realistic, corse-grained protein models with various degree of frustration in the protein energy landscape. We also investigate the efficacy of using several previously proposed optimal setups, such as the highest temperature, in REXMD. At the end, we also propose a simple strategy to circumvent the phase space trapping by periodically forcing replicas to visit different temperature ranges.

Many human diseases, including episodic ataxia, diabetes, epilepsy, cystic fibrosis and Alzheimer's dementia, are related to defective ion channels. A series of channel forming peptides derived from the second transmembran domain of the α1-subunit of the glycine receptor (M2GlyR) have been designed by the Tomich lab with improved anion conduction rate and aqueous solubility. To rationally understand the physiological properties of these synthetic channels and to identify improved designs, we combine NMR, biophysical data, and molecular modeling to provide a structural basis for understanding key physiochemical properties that govern the chloride conductivity and selectivity. Initial structural models were first constructed for one of our lead design, p22-T19R/S22W (KKKKP ARVGL GITTV LTMRT QW), primarily based on the monomer structure from solution NMR, amphipathicity consideration, and the oligomeric state of the channel assembly. Long molecular dynamic simulations in explicit membrane and water were then carried out to characterize the channel structural and dynamic properties. Interestingly, independent simulations from initial constructs with different handiness of helix packing (left, straight, and right) all converge to a similar structural ensemble with left-handed helix assembly. The predicted pore-lining residues are also in excellent agreement with a previous set of cysteine-scanning experiments. Coupled with parallel experimental characterizations in the Tomich lab, the simulation provides important insights into the structural basis of the activity of these synthetic channels.

Intrinsically disordered proteins (IDPs) are functional proteins where a lack of stable tertiary structures is required for function. Many of the IDPs involved in cellular regulation and signaling have substantial residual structures in the unbound state and fold into stable structures upon binding to their biological partners. Specific roles of these residual structures in and the underlying mechanisms of coupled binding and folding are poorly understood. Here we use physics-based atomistic simulations to compute the multidimensional free energy surfaces of coupled folding and binding of the intrinsically disordered p53 extreme C-terminus to protein S100B(betabeta). The results show that, even though the unbound p53 peptide appears to sample several alternative folded states previously observed when in complex with various targets, it binds to S100B(betabeta) through formation of nonspecific complexes, i.e., a "fly-casting"-like process. The current work, together with previous NMR and coarse-grained modeling studies of another prototypical system, suggests that the main role of the residual structures in the unbound states of regulatory IDPs might be to provide thermodynamic control of binding through modulating the entropic cost of folding and not to enhance the binding rate by acting as initial contact sites.

Intrinsically disordered proteins (IDP) can exist as ensembles of disordered conformations under physiological conditions, and such intrinsic disorder often plays important roles in their functions. The kinase-inducible transactivation domain (KID) from cAMP-response element-binding protein (CREB) has distinct ordered structure with its binding partner KIX, but is mostly unstructured in unbound state. The phosphorylation on Ser133 residue of KID increases the binding potency of the peptide toward KIX, but its impacts the disordered states remain unclear. We have carried out atomistic simulations in an implicit solvent to study effects of above-mentioned phosphorylation on the structure of unbound KID peptide. The results reveal that while the phosphorylation does not affect the average residue helicities, but has importance consequences on the flexibility of the peptide as well as the length and population of the transient helical segments. In particular, phosphorylation appears to restrict the accessible conformational space of the loop connecting two helices, and reduces the entropic penalty of folding upon binding. This entropic contribution, estimated to be ∼1.5R from 4D joint backbone torsion distributions of Arg130 and Arg131 residues of KID, supplements the salt-bridges between pSer133 of KID and Lys662 and Tyr658 residues of KIX. This effect was not previously recognized due to inaccessibility of the structural details of the disordered ensembles from experiments. Success of these simulations is very encouraging, and demonstrates the feasibility of an implicit solvent-based computational framework for accurate atomistic simulation of IDPs.