Principles of Alternating Access in Multidrug and Toxin Extrusion (MATE) Transporters

The multidrug and toxin extrusion (MATE) transporters catalyze active efflux of a broad range of chemically- and structurally-diverse compounds including antimicrobials and chemotherapeutics, thus contributing to multidrug resistance in pathogenic bacteria and cancers. Multiple methodological approaches have been taken to investigate the structural basis of energy transduction and substrate translocation in MATE transporters. Crystal structures representing members from all three MATE subfamilies have been interpreted within the context of an alternating access mechanism that postulates occupation of distinct structural intermediates in a conformational cycle powered by electrochemical ion gradients. Here we review the structural biology of MATE transporters, integrating the crystallographic models with biophysical and computational studies to define the molecular determinants that shape the transport energy landscape. This holistic analysis highlights both shared and disparate structural and functional features within the MATE family, which underpin an emerging theme of mechanistic diversity within the framework of a conserved structural scaffold.     

Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer

Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.     

Activation and Speciation Mechanisms in Class A GPCRs

Accurate development of allosteric modulators of GPCRs require a thorough assessment of their sequence, structure, and dynamics, toward gaining insights into their mechanisms of actions shared by family members, as well as dynamic features that distinguish subfamilies. Building on recent progress in the characterization of the signature dynamics of proteins, we analyzed here a dataset of 160 Class A GPCRs to determine their sequence similarities, structural landscape, and dynamic features across different species (human, bovine, mouse, squid, and rat), different activation states (active/inactive), and different subfamilies. The two dominant directions of variability across experimentally resolved structures, identified by principal component analysis of the dataset, shed light to cooperative mechanisms of activation, subfamily differentiation, and speciation of Class A GPCRs. The analysis reveals the functional significance of the conformational flexibilities of specific structural elements, including: the dominant role of the intracellular loop 3 (ICL3) together with the cytoplasmic ends of the adjoining helices TM5 and TM6 in enabling allosteric activation; the role of particular structural motifs at the extracellular loop 2 (ECL2) connecting TM4 and TM5 in binding ligands specific to different subfamilies; or even the differentiation of the N-terminal conformation across different species. Detailed analyses of the modes of motions accessible to the members of the dataset and their variations across members demonstrate how the active and inactive states of GPCRs obey distinct conformational dynamics. The collective fluctuations of the GPCRs are robustly defined in the active state, while the inactive conformers exhibit broad variance among members.     

The Trimeric Major Capsid Protein of Mavirus is stabilized by its Interlocked N-termini Enabling Core Flexibility for Capsid Assembly

Icosahedral viral capsids assemble with high fidelity from a large number of identical buildings blocks. The mechanisms that enable individual capsid proteins to form stable oligomeric units (capsomers) while affording structural adaptability required for further assembly into capsids are mostly unknown.Understanding these mechanisms requires knowledge of the capsomers’ dynamics, especially for viruses where no additional helper proteins are needed during capsid assembly like for the Mavirus virophage that despite its complexity (triangulation number T = 27) can assemble from its major capsid protein (MCP) alone. This protein forms the basic building block of the capsid namely a trimer (MCP₃) of double-jelly roll protomers with highly intertwined N-terminal arms of each protomer wrapping around the other two at the base of the capsomer, secured by a clasp that is formed by part of the C-terminus.Probing the dynamics of the capsomer with HDX mass spectrometry we observed differences in conformational flexibility between functional elements of the MCP trimer. While the N-terminal arm and clasp regions show above average deuterium incorporation, the two jelly-roll units in each protomer also differ in their structural plasticity, which might be needed for efficient assembly. Assessing the role of the N-terminal arm in maintaining capsomer stability showed that its detachment is required for capsomer dissociation, constituting a barrier towards capsomer monomerisation. Surprisingly, capsomer dissociation was irreversible since it was followed by a global structural rearrangement of the protomers as indicated by computational studies showing a rearrangement of the N-terminus blocking part of the capsomer forming interface.     

Conformational Dynamics Allows Sampling of an “Active-like” State by Oncogenic K-Ras-GDP

Mutations in K-Ras GTPase replacing Gly12 with either Asp or Val are common in cancer. These mutations decelerate intrinsic and catalyzed GTP hydrolysis, leading to accumulation of K-Ras-GTP in cells. Signaling cascades initiated by K-Ras-GTP promote cell proliferation, survival, and invasion. Despite functional differences between the most frequent G12D mutation and the most aggressive and chemotherapy resistant G12V mutation, their long-suspected distinct structural features remain elusive. Using NMR, X-ray structures, and computational methods, we found that oncogenic mutants of K-Ras4B, the predominant splice variant of K-Ras, exhibit distinct conformational dynamics when GDP-bound, visiting the “active-like” conformational state similar to the one observed in GTP-bound K-Ras. This behavior distinguishes G12V from wild type and G12D K-Ras4B-GDP. The likely reason is interactions between the aliphatic sidechain of V12 and the Switch II region of K-Ras4B^G12V-GDP, which are distinct in K-Ras4B^G12D-GDP. In the X-ray structures, crystal contacts reduce the dynamics of the sidechain at position 12 by stabilizing the Switch I region of the protein. This explains why structural differences between G12V and G12D K-Ras have yet not been reported. Together, our results suggest a previously unknown mechanism of K-Ras activation. This mechanism relies on conformational dynamics caused by specific oncogenic mutations in the GDP-bound state. Our findings also imply that the therapeutic strategies decreasing the level of K-Ras-GTP by interfering with nucleotide exchange or by expediting GTP hydrolysis may work differently in different oncogenic mutants.     

Conformational Transitions in Yeast Chorismate Mutase Important for Allosteric Regulation as Identified by Nuclear Magnetic Resonance Spectroscopy

Proteins fluctuate between different conformations in solution, and these conformational fluctuations can be important for protein function and allosteric regulation. The chorismate mutase from Saccharomyces cerevisiae (ScCM), a key enzyme in the biosynthesis of aromatic amino acids, is allosterically activated and inhibited by tryptophan and tyrosine, respectively. It was initially proposed that in the absence of effector, ScCM fluctuates between activated R and inhibited T conformations according to the Monod-Wyman-Changeux (MWC) model, although a more complex regulation pattern was later suggested by mutagenesis and kinetic data. Here we used NMR relaxation dispersion experiments to understand the conformational fluctuations on the microsecond-to-millisecond timescale that occur in ScCM. In the absence of allosteric effectors, ScCM did not exclusively exchange between T and R conformations, suggesting that the two-state MWC model is insufficient to explain conformational dynamics. Addition of tyrosine led to the quenching of much of the motion on this timescale, while new motions were identified in the presence of tryptophan. These new motions are consistent with conformational fluctuations into an alternative conformation that may be important for enzyme activity.     

Conformational analysis by NMR and molecular dynamics of adamantane-doxorubicin prodrugs and their assemblies with β-cyclodextrin: A focus on the design of platforms for controlled drug delivery

Nanoscale design and construction of affinity-based drug delivery systems (ADDS) is an active research area with enormous potential for the improvement of cancer treatment. For the therapeutic load of these ADDS, a promising strategy is the design of pH-sensitive prodrugs based on the construction of conjugates between adamantane and doxorubicin (Ad-Dox), which stands out as an excellent model system to obtain novel supramolecular materials. Construction of these prodrugs involves a modification of three zones of doxorubicin which in principle does not affect the action mechanism: the carbonyl group C13 (hydrazone linker), the primary alcohol neighboring the carbonyl (ester linker) and the 3′ amino group of daunosamine sugar (amide linker). These modifications are aimed to improve the efficacy and reduce the systemic toxicity of the drug chemotherapy by controlling its release in cancer cells. In this work, we performed 2D NMR experiments and molecular dynamics simulations to characterize the conformational changes of three constructed prodrugs. Our results demonstrated that ring A and the daunsamine sugar of the hydrazone and amide linkers conserve the half-chair state ⁹H₈, while the ester linker disrupts this conformation. Our study also showed that the hydrazone-linked compound (Ad-h-Dox) does not modify the conformation of the original drug and maintains cytotoxic activity. Moreover, the inclusion complex (IC) of Ad-h-Dox with β-cyclodextrin (βCD) generated a highly soluble platform in water, whereas the ester-linked compound (Ad-e-Dox) causes the loss of biological activity. This study proves that Ad-h-Dox prodrug can be an optimum prodrug and act as a building block for a more complex drug transport system.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Elephants in the Dark: Insights and Incongruities in Pentameric Ligand-gated Ion Channel Models

The superfamily of pentameric ligand-gated ion channels (pLGICs) comprises key players in electrochemical signal transduction across evolution, including historic model systems for receptor allostery and targets for drug development. Accordingly, structural studies of these channels have steadily increased, and now approach 250 depositions in the protein data bank. This review contextualizes currently available structures in the pLGIC family, focusing on morphology, ligand binding, and gating in three model subfamilies: the prokaryotic channel GLIC, the cation-selective nicotinic acetylcholine receptor, and the anion-selective glycine receptor. Common themes include the challenging process of capturing and annotating channels in distinct functional states; partially conserved gating mechanisms, including remodeling at the extracellular/transmembrane-domain interface; and diversity beyond the protein level, arising from posttranslational modifications, ligands, lipids, and signaling partners. Interpreting pLGIC structures can be compared to describing an elephant in the dark, relying on touch alone to comprehend the many parts of a monumental beast: each structure represents a snapshot in time under specific experimental conditions, which must be integrated with further structure, function, and simulations data to build a comprehensive model, and understand how one channel may fundamentally differ from another.     

Ceramide-1-phosphate transfer protein promotes sphingolipid reorientation needed for binding during membrane interaction

Lipid transfer proteins acquire and release their lipid cargoes by interacting transiently with source and destination biomembranes. In the GlycoLipid Transfer Protein (GLTP) superfamily, the two-layer all-α-helical GLTP-fold defines proteins that specifically target sphingolipids (SLs) containing either sugar or phosphate headgroups via their conserved but evolutionarily-modified SL recognitions centers. Despite comprehensive structural insights provided by X-ray crystallography, the conformational dynamics associated with membrane interaction and SL uptake/release by GLTP superfamily members have remained unknown. Herein, we report insights gained from molecular dynamics (MD) simulations into the conformational dynamics that enable ceramide-1-phosphate transfer proteins (CPTPs) to acquire and deliver ceramide-1-phosphate (C1P) during interaction with 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. The focus on CPTP reflects this protein’s involvement in regulating pro-inflammatory eicosanoid production and autophagy-dependent inflammasome assembly that drives interleukin (IL-1β and IL-18) production and release by surveillance cells. We found that membrane penetration by CPTP involved α-6 helix and the α-2 helix N-terminal region, was confined to one bilayer leaflet, and was relatively shallow. Large-scale dynamic conformational changes were minimal for CPTP during membrane interaction or C1P uptake except for the α-3/α-4 helices connecting loop, which is located near the membrane interface and interacts with certain phosphoinositide headgroups. Apart from functioning as a shallow membrane-docking element, α-6 helix was found to adeptly reorient membrane lipids to help guide C1P hydrocarbon chain insertion into the interior hydrophobic pocket of the SL binding site.These findings support a proposed ‘hydrocarbon chain-first’ mechanism for C1P uptake, in contrast to the ‘lipid polar headgroup-first’ uptake used by most lipid-transfer proteins.     

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.     

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.     

The role of amino acid metabolism alterations in pancreatic cancer: From mechanism to application

The incidence of pancreatic cancer is increasing in both developed and developing Nations. In recent years, various research evidence suggested that reprogrammed metabolism may play a key role in pancreatic cancer tumorigenesis and development. Therefore, it has great potential as a diagnostic, prognostic and therapeutic target. Amino acid metabolism is deregulated in pancreatic cancer, and changes in amino acid metabolism can affect cancer cell status, systemic metabolism in malignant tumor patients and mistakenly involved in different biological processes including stemness, proliferation and growth, invasion and migration, redox state maintenance, autophagy, apoptosis and even tumor microenvironment interaction. Generally, the above effects are achieved through two pathways, energy metabolism and signal transduction. This review aims to highlight the current research progress on the abnormal alterations of amino acids metabolism in pancreatic cancer, how they affect tumorigenesis and development of pancreatic cancer and the application prospects of them as diagnostic, prognostic and therapeutic targets.     

Intradimeric Walker A ATPases: Conserved Features of A Functionally Diverse Family

Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis. Although the proteins in this subset have vastly differing functions, ranging from electron transport during nitrogen fixation to targeting of integral membrane proteins to their correct membranes, they have evolved from a shared ancestor and have thus retained common structural features that affect their functions. These commonalities have only been disparately characterized in the context of their individual proteins systems, but have not been generally annotated as features that unite the members of this family. In this review, we report an analysis based on the sequences, structures, and functions of several members in this family that highlight their remarkable similarities. A principal feature of these proteins is their dependence on homodimerization. Since their functionalities are heavily influenced by changes that happen in conserved elements at the dimer interface, we refer to the members of this subclass as intradimeric Walker A ATPases.     

Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by γ-Secretase

The intramembrane protease γ-secretase activates important signaling molecules, such as Notch receptors. It is still unclear, however, how different elements within the primary structure of substrate transmembrane domains (TMDs) contribute to their cleavability. Using a newly developed yeast-based cleavage assay, we identified three crucial regions within the TMDs of the paralogs Notch1 and Notch3 by mutational and gain-of-function approaches. The AAAA or AGAV motifs within the N-terminal half of the TMDs were found to confer strong conformational flexibility to these TMD helices, as determined by mutagenesis coupled to deuterium/hydrogen exchange. Crucial amino acids within the C-terminal half may support substrate docking into the catalytic cleft of presenilin, the enzymatic subunit of γ-secretase. Further, residues close to the C-termini of the TMDs may stabilize a tripartite β-sheet in the substrate/enzyme complex. NMR structures reveal different extents of helix bending as well as an ability to adopt widely differing conformational substates, depending on the sequence of the N-terminal half. The difference in cleavability between Notch1 and Notch3 TMDs is jointly determined by the conformational repertoires of the TMD helices and the sequences of the C-terminal half, as suggested by mutagenesis and building molecular models. In sum, cleavability of a γ-secretase substrate is enabled by different functions of cooperating TMD regions, which deepens our mechanistic understanding of substrate/non-substrate discrimination in intramembrane proteolysis.