Quantum-Engineered Devices Based on 2D Materials for Next-Generation Information Processing and Storage.

As an approximation to the quantum state of solids, the band theory, developed nearly seven decades ago, fostered the advance of modern integrated solid-state electronics, one of the most successful technologies in the history of human civilization. Nonetheless, their rapidly growing energy consumption and accompanied environmental issues call for more energy-efficient electronics and optoelectronics, which necessitate the exploration of more advanced quantum mechanical effects, such as band-to-band tunneling, spin-orbit coupling, spin-valley locking, and quantum entanglement. The emerging 2D layered materials, featured by their exotic electrical, magnetic, optical, and structural properties, provide a revolutionary low-dimensional and manufacture-friendly platform (and many more opportunities) to implement these quantum-engineered devices, compared to the traditional electronic materials system. Here, the progress in quantum-engineered devices is reviewed and the opportunities/challenges of exploiting 2D materials are analyzed to highlight their unique quantum properties that enable novel energy-efficient devices, and useful insights to quantum device engineers and 2D-material scientists are provided.

A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl-/H+ transport cycle.

Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl- for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl- substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure provides evidence for a unified model of H+/Cl- transport that reconciles existing data on all CLC-type proteins.

Cells are shielded from harmful molecules and other threats by a thin, flexible layer called the membrane. However, this barrier also prevents chloride, sodium, protons and other ions from moving in or out of the cell. Channels and transporters are two types of membrane proteins that form passageways for these charged particles. Channels let ions flow freely from one side of the membrane to the other. To do so, these proteins change their three-dimensional shape to open or close as needed. On the other hand, transporters actively pump ions across the membrane to allow the charged particles to accumulate on one side. The shape changes needed for that type of movement are different: the transporters have to open a passageway on one side of the membrane while closing it on the other side, alternating openings to one side or the other. In general, channels and transporters are not related to each other, but one exception is a group called CLCs proteins. Present in many organisms, this family contains a mixture of channels and transporters. For example, humans have nine CLC proteins: four are channels that allow chloride ions in and out, and five are 'exchange transporters' that make protons and chloride ions cross the membrane in opposite directions. These proteins let one type of charged particle move freely across the membrane, which generates energy that the transporter then uses to actively pump the other ion in the direction needed by the cell. Yet, the exact three-dimensional changes required for CLC transporters and channels to perform their roles are still unknown. To investigate this question, Chavan, Cheng et al. harnessed a technique called X-ray crystallography, which allows scientists to look at biological molecules at the level of the atom. This was paired with other methods to examine a CLC mutant that adopts the shape of a normal CLC transporter when it is loaded with a proton. The experiments revealed how various elements in the transporter move relative to each other to adopt a structure that allows protons and chloride ions to enter the protein from opposite sides of the membrane, using separate pathways. While obtained on a bacterial CLC, these results can be applied to other CLC channels and transporters (including those in humans), shedding light on how this family transports charged particles across membranes. From bone diseases to certain types of seizures, many human conditions are associated with poorly functioning CLCs. Understanding the way these structures change their shapes to perform their roles could help to design new therapies for these health problems.

A selective class of inhibitors for the CLC-Ka chloride ion channel.

CLC proteins are a ubiquitously expressed family of chloride-selective ion channels and transporters. A dearth of pharmacological tools for modulating CLC gating and ion conduction limits investigations aimed at understanding CLC structure/function and physiology. Herein, we describe the design, synthesis, and evaluation of a collection of N-arylated benzimidazole derivatives (BIMs), one of which (BIM1) shows unparalleled (>20-fold) selectivity for CLC-Ka over CLC-Kb, the two most closely related human CLC homologs. Computational docking to a CLC-Ka homology model has identified a BIM1 binding site on the extracellular face of the protein near the chloride permeation pathway in a region previously identified as a binding site for other less selective inhibitors. Results from site-directed mutagenesis experiments are consistent with predictions of this docking model. The residue at position 68 is 1 of only ∼20 extracellular residues that differ between CLC-Ka and CLC-Kb. Mutation of this residue in CLC-Ka and CLC-Kb (N68D and D68N, respectively) reverses the preference of BIM1 for CLC-Ka over CLC-Kb, thus showing the critical role of residue 68 in establishing BIM1 selectivity. Molecular docking studies together with results from structure-activity relationship studies with 19 BIM derivatives give insight into the increased selectivity of BIM1 compared with other inhibitors and identify strategies for further developing this class of compounds.

Flexible-body motions of calmodulin and the farnesylated hypervariable region yield a high-affinity interaction enabling K-Ras4B membrane extraction.

In calmodulin (CaM)-rich environments, oncogenic KRAS plays a critical role in adenocarcinomas by promoting PI3K/Akt signaling. We previously proposed that at elevated calcium levels in cancer, CaM recruits PI3Kα to the membrane and extracts K-Ras4B from the membrane, organizing a K-Ras4B-CaM-PI3Kα ternary complex. CaM can thereby replace a missing receptor-tyrosine kinase signal to fully activate PI3Kα. Recent experimental data show that CaM selectively promotes K-Ras signaling but not of N-Ras or H-Ras. How CaM specifically targets K-Ras and how it extracts it from the membrane in KRAS-driven cancer is unclear. Obtaining detailed structural information for a CaM-K-Ras complex is still challenging. Here, using molecular dynamics simulations and fluorescence experiments, we observed that CaM preferentially binds unfolded K-Ras4B hypervariable regions (HVRs) and not α-helical HVRs. The interaction involved all three CaM domains including the central linker and both lobes. CaM specifically targeted the highly polybasic anchor region of the K-Ras4B HVR that stably wraps around CaM's acidic linker. The docking of the farnesyl group to the hydrophobic pockets located at both CaM lobes further enhanced CaM-HVR complex stability. Both CaM and K-Ras4B HVR are highly flexible molecules, suggesting that their interactions permit highly dynamic flexible-body motions. We, therefore, anticipate that the flexible-body interaction is required to extract K-Ras4B from the membrane, as conformational plasticity enables CaM to orient efficiently to the polybasic HVR anchor, which is partially diffused into the liquid-phase membrane. Our structural model of the CaM-K-Ras4B HVR association provides plausible clues to CaM's regulatory action in PI3Kα activation involving the ternary complex in cell proliferation signaling by oncogenic K-Ras.

The higher level of complexity of K-Ras4B activation at the membrane.

Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.-Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane.

Revealing an outward-facing open conformational state in a CLC Cl(-)/H(+) exchange transporter.

CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as [H(+)] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.

High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site.

Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible.

GTP-Dependent K-Ras Dimerization.

Ras proteins recruit and activate effectors, including Raf, that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. It has been suspected that dimeric Ras may promote dimerization and activation of Raf. Here, we show that the GTP-bound catalytic domain of K-Ras4B, a highly oncogenic splice variant of the K-Ras isoform, forms stable homodimers. We observe two major dimer interfaces. The first, highly populated ß-sheet dimer interface is at the Switch I and effector binding regions, overlapping the binding surfaces of Raf, PI3K, RalGDS, and additional effectors. This interface has to be inhibitory to such effectors. The second, helical interface also overlaps the binding sites of some effectors. This interface may promote activation of Raf. Our data reveal how Ras self-association can regulate effector binding and activity, and suggest that disruption of the helical dimer interface by drugs may abate Raf signaling in cancer.

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function.

Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α1-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α1A/B-AR and CXCR4, and inhibited Ca(2+) mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α1-AR activation. CXCR4 silencing reduced α1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca(2+) fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC50 of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α1-AR-mediated function in VSMC and that activation of CXCR4 enhances the potency of α1-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α1-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.

Mechanisms of membrane binding of small GTPase K-Ras4B farnesylated hypervariable region.

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling.

Plasma membrane regulates Ras signaling networks.

Ras GTPases activate more than 20 signaling pathways, regulating such essential cellular functions as proliferation, survival, and migration. How Ras proteins control their signaling diversity is still a mystery. Several pieces of evidence suggest that the plasma membrane plays a critical role. Among these are: (1) selective recruitment of Ras and its effectors to particular localities allowing access to Ras regulators and effectors; (2) specific membrane-induced conformational changes promoting Ras functional diversity; and (3) oligomerization of membrane-anchored Ras to recruit and activate Raf. Taken together, the membrane does not only attract and retain Ras but also is a key regulator of Ras signaling. This can already be gleaned from the large variability in the sequences of Ras membrane targeting domains, suggesting that localization, environment and orientation are important factors in optimizing the function of Ras isoforms.

A novel method for the production of fully modified K-Ras 4B.

Post-translational modifications in proteins play a major functional role. Post-translational modifications affect the way proteins interact with each other, bind nucleotides, and localize in cellular compartments. Given the importance of post-translational modifications in protein biology, development of methods to produce post-translationally modified proteins for biochemical and biophysical studies is timely and significant. At the same time, obtaining post-translationally modified proteins in bacterial expression systems is often problematic. Here, we describe a novel recombinant approach to prepare human K-Ras 4B, a protein that is post-translationally farnesylated, proteolytically cleaved, and methylated in its C-terminus. K-Ras 4B is a member of the Ras subfamily of small GTPases and is of interest because it is frequently mutated in human cancer. The method relies on separate production of two structural domains-the N-terminal catalytic domain and the C-terminal peptide chemically modified with S-farnesyl-L-cysteine methyl ester. After the two domains are prepared, they are ligated together using the transpeptidase enzyme, sortase. Our procedure starts with the use of the plasmid of K-Ras 4B catalytic domain containing the sortase recognition sequence. After this, we describe the bacterial expression and purification steps used to purify K-Ras 4B and the preparation of the conjugated C-terminal peptide. The procedure ends with the sortase-mediated ligation technique. The produced post-translationally modified K-Ras 4B is active in a number of assays, including a GTP hydrolysis assay, Raf-1 binding assay, and surface plasmon resonance-based phospholipid binding assay.

Application of reductive ¹³C-methylation of lysines to enhance the sensitivity of conventional NMR methods.

NMR is commonly used to investigate macromolecular interactions. However, sensitivity problems hamper its use for studying such interactions at low physiologically relevant concentrations. At high concentrations, proteins or peptides tend to aggregate. In order to overcome this problem, we make use of reductive ¹³C-methylation to study protein interactions at low micromolar concentrations. Methyl groups in dimethyl lysines are degenerate with one ¹³CH3 signal arising from two carbons and six protons, as compared to one carbon and three protons in aliphatic amino acids. The improved sensitivity allows us to study protein-protein or protein-peptide interactions at very low micromolar concentrations. We demonstrate the utility of this method by studying the interaction between the post-translationally lipidated hypervariable region of a human proto-oncogenic GTPase K-Ras and a calcium sensor protein calmodulin. Calmodulin specifically binds K-Ras and modulates its downstream signaling. This binding specificity is attributed to the unique lipidated hypervariable region of K-Ras. At low micromolar concentrations, the post-translationally modified hypervariable region of K-Ras aggregates and binds calmodulin in a non-specific manner, hence conventional NMR techniques cannot be used for studying this interaction, however, upon reductively methylating the lysines of calmodulin, we detected signals of the lipidated hypervariable region of K-Ras at physiologically relevant nanomolar concentrations. Thus, we utilize ¹³C-reductive methylation of lysines to enhance the sensitivity of conventional NMR methods for studying protein interactions at low concentrations.

ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H2O2. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.