Relating nucleotide‐dependent conformational changes in free tubulin dimers to tubulin assembly

The complex dynamic behavior of microtubules (MTs) is believed to be primarily due to the αβ‐tubulin dimer architecture and its intrinsic GTPase activity. Hence, a detailed knowledge of the conformational variations of isolated α‐GTP‐β‐GTP‐ and α‐GTP‐β‐GDP‐tubulin dimers in solution and their implications to interdimer interactions and stability is directly relevant to understand the MT dynamics. An attempt has been made here by combining molecular dynamics (MD) simulations and protein–protein docking studies that unravels key structural features of tubulin dimer in different nucleotide states and correlates their association to tubulin assembly. Results from simulations suggest that tubulin dimers and oligomers attain curved conformations in both GTP and GDP states. Results also indicate that the tubulin C‐terminal domain and the nucleotide state are closely linked. Protein–protein docking in combination with MD simulations suggest that the GTP‐tubulin dimers engage in relatively stronger interdimer interactions even though the interdimer interfaces are bent in both GTP and GDP tubulin complexes, providing valuable insights on in vitro finding that GTP‐tubulin is a better assembly candidate than GDP‐tubulin during the MT nucleation and elongation processes. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 282–291, 2013.     

Assembly of α-synuclein aggregates on phospholipid bilayers

The spontaneous self-assembly of α-synuclein (α-syn) into aggregates of different morphologies is associated with the development of Parkinson's disease. However, the mechanism behind the spontaneous assembly remains elusive. The current study shows a novel effect of phospholipid bilayers on the assembly of the α-syn aggregates. Using time-lapse atomic force microscopy, it was discovered that α-syn assembles into aggregates on bilayer surfaces, even at the nanomolar concentration range. The efficiency of the aggregation process depends on the membrane composition, with the greatest efficiency observed for of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS). Importantly, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation is a mechanism by which pathological aggregates may be produced. Computational modeling revealed that dimers of α-syn assembled rapidly, through the membrane-bound monomer on POPS bilayer, due to an aggregation-prone orientation of α-syn. Interaction of α-syn with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) leads to a binding mode that does not induce a fast assembly of the dimer. Based on these findings, we propose a model in which the interaction of α-syn with membranes plays a critical role initiating the formation of α-syn aggregates and the overall aggregation process.     

Comparative study of stability and transport of molecules through cyclic peptide nanotube and aquaporin: a molecular dynamics simulation approach

Abstract

The structural stability and transport properties of the cyclic peptide nanotube (CPN) 8?×?[Cys–Gly–Met–Gly]₂ in different phospholipid bilayers such as POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) and POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine) with water have been investigated using molecular dynamics (MD) simulation. The hydrogen bonds and non-bonded interaction energies were calculated to study the stability in different bilayers. One µs MD simulation in POPA lipid membrane reveals the stability of the cyclic peptide nanotube, and the simulations at various temperatures manifest the higher stability of 8?×?[Cys–Gly–Met–Gly]₂. We demonstrated that the presence of sulphur-containing amino acids in CPN enhances the stability through disulphide bonds between the adjacent rings. Further, the water permeation coefficient of the CPN is calculated and compared with human aquaporin-2 (AQP2) channel protein. It is found that the coefficients are highly comparable to the AQP2 channel though the mechanism of water transport is not similar to AQP 2; the flow of water in the CPN is taking place as a two-line 1–2–1–2 file fashion. In addition to that, the transport behavior of Na⁺ and K⁺ ions, single water molecule, urea and anti-cancer drug fluorouracil were investigated using pulling simulation and potential of mean force calculation. The above transport behavior shows that Na⁺ is trapped in CPN for a longer time than other molecules. Also, the interactions of the ions and molecules in Cα and mid-Cα plane were studied to understand the transport behavior of the CPN. Abbreviations AQP2 Aquaporin-2

CPN Cyclic peptide nanotube

MD Molecular dynamics

POPA 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid

POPE 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

POPG 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol

POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine

Communicated by Ramaswamy H. Sarma     

G-Domain Dimerization Orchestrates the tRNA Wobble Modification Reaction in the MnmE/GidA Complex

MnmE and GidA are involved in the modification of wobble uridine to carboxymethylaminomethyl uridine in certain tRNAs. Malfunctioning of the human orthologs has been implicated in mitochondrial diseases. MnmE is a conserved G protein activated by dimerization. Here, we show that complex formation between MnmE and GidA involves large conformational changes that induce G-domain dimerization of MmnE and that GidA co-stimulates GTP hydrolysis on MnmE. Starting from a structural model of the complex, we identify interface mutations disrupting complex formation or communication. Although GidA does not directly contact the G-domains, conformational changes in MnmE, induced by G-domain dimerization in the triphosphate state, regulate the affinity for GidA. We developed a tRNA modification assay and demonstrate for the first time in vitro that the MnmE/GidA complex catalyzes incorporation of glycine into tRNA. An intact MnmE/GidA complex rather than their sequential action is crucial for in vitro modification. Since only GTP, but not GDP or non-hydrolyzable GTP analogs, drives the MnmE/GidA-catalyzed modification reaction, we conclude that GTP hydrolysis is essential for activity. We finally show that an active GTPase, an intact MnmE/GidA communication, and dimerization of G-domains are necessary for in vivo functioning since mutations disrupting either result in a respiratory deficient phenotype in yeast.     

The role of G-domain orientation and nucleotide state on the Ras isoform-specific membrane interaction

Ras proteins are proto-oncogenes that function as molecular switches linking extracellular stimuli with an overlapping but distinctive range of biological outcomes. Although modulatable interactions between the membrane and the Ras C-terminal hypervariable region (HVR) harbouring the membrane anchor motifs enable signalling specificity to be determined by their location, it is becoming clear that the spatial orientation of different Ras proteins is also crucial for their functions. To reveal the orientation of the G-domain at membranes, we conducted an extensive study on different Ras isoforms anchored to model raft membranes. The results show that the G-domain mediates the Ras–membrane interaction by inducing different sets of preferred orientations in the active and inactive states with largely parallel orientation relative to the membrane of most of the helices. The distinct locations of the different isoforms, exposing them to different effectors and regulators, coupled with different G-domain-membrane orientation, suggests synergy between this type of recognition motif and the specificity conferred by the HVR, thereby validating the concept of isoform specificity in Ras.     

Characterization of the homodimerization interface and functional hotspots of the CXCR4 chemokine receptor

The recent crystallographic structures of the human chemokine CXC Receptor 4 (CXCR4) provide experimental evidence of a human G Protein-Coupled Receptor (GPCR) dimer in atomic detail. The CXCR4 homodimers reveal an unexpected dimerization mode involving transmembrane helices TM5 and TM6, which is examined here using all-atom molecular dynamics (MD) simulations in the physiological environment of a lipid bilayer. The bacteriophage T4 lysozyme (T4L), which was fused to the crystallized protein but absent in our simulations, is found to slightly affect the observed relative position of the protomers in the two dimers studied here, and consequently some rearrangements of the dimerization interface are proposed. In addition, the simulations provide further evidence about the role of the two stabilizing single point mutations introduced to crystallize the receptor. Finally, this work analyzes the structural and dynamic role of key residues involved both in ligand binding and in the infection process of HIV. In particular, the different side chain conformations of His113(3.39) are found to influence the dynamics of the surrounding functional hotspot region being evaluated both in the presence and in the absence of the co-crystallized ligand IT1t. The analysis reported here adds valuable knowledge for future structure-based drug design (SBDD) efforts on this pharmacological target.     

Structure and Function of the FeoB G-Domain from Methanococcus jannaschii

FeoB in bacteria and archaea is involved in the uptake of ferrous iron (Fe²⁺), an important cofactor in biological electron transfer and catalysis. Unlike any other known prokaryotic membrane protein, FeoB contains a GTP-binding domain at its N-terminus. We determined high-resolution X-ray structures of the FeoB G-domain from Methanococcus jannaschii with and without bound GDP or Mg²⁺-GppNHp. The G-domain forms the same dimer in all three structures, with the nucleotide-binding pockets at the dimer interface, as in the ATP-binding domain of ABC transporters. The G-domain follows the typical fold of nucleotide-binding proteins, with a β-strand inserted in switch I that becomes partially disordered upon GTP binding. Switch II does not contact the nucleotide directly and does not change its conformation in response to the bound nucleotide. Release of the nucleotide causes a rearrangement of loop L6, which we identified as the G5 region of FeoB. Together with the C-terminal helix, this loop may transmit the information about the nucleotide-bound state from the G-domain to the transmembrane region of FeoB.     

Simultaneous formation of right- and left-handed anti-parallel coiled-coil interfaces by a coil2 fragment of human lamin A

The elementary building block of all intermediate filaments (IFs) is a dimer featuring a central α-helical rod domain flanked by the N- and C-terminal end domains. In nuclear IF proteins (lamins), the rod domain consists of two coiled-coil segments, coil1 and coil2, that are connected by a short non-helical linker. Coil1 and the C-terminal part of coil2 contain the two highly conserved IF consensus motifs involved in the longitudinal assembly of dimers. The previously solved crystal structure of a lamin A fragment (residues 305-387) corresponding to the second half of coil2 has yielded a parallel left-handed coiled coil. Here, we present the crystal structure and solution properties of another human lamin A fragment (residues 328-398), which is largely overlapping with fragment 305-387 but harbors a short segment of the tail domain. Unexpectedly, no parallel coiled coil forms within the crystal. Instead, the α-helices are arranged such that two anti-parallel coiled-coil interfaces are formed. The most significant interface has a right-handed geometry, which is accounted for by a characteristic 15-residue repeat pattern that overlays with the canonical heptad repeat pattern. The second interface is a left-handed anti-parallel coiled coil based on the predicted heptad repeat pattern. In solution, the fragment reveals only a weak dimerization propensity. We speculate that the C-terminus of coil2 might unzip, thereby allowing for a right-handed coiled-coil interface to form between two laterally aligned dimers. Such an interface might co-exist with a heterotetrameric left-handed coiled-coil assembly, which is expected to be responsible for the longitudinal A_CN contact.     

Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase dependent

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that, for a dipalmitoylphosphatidylcholine (DPPC) membrane whose gel-to-fluid lipid phase transition occurs at 314 K, while the TEMPO spin label is stabilized above the bilayer interface in the gel phase, there is a preferential location of TEMPO below the membrane interface in the fluid phase. For bilayers made of unsaturated lipids, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of the TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.     

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and P_i release can be uncoupled and that G-domain dissociation is directly responsible for the ‘ON’ state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and P_i. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.     

N-Ras Forms Dimers at POPC Membranes

Ras is a central regulator of cellular signaling pathways. It is mutated in 20–30% of human tumors. To perform its function, Ras has to be bound to a membrane by a posttranslationally attached lipid anchor. Surprisingly, we identified here dimerization of membrane anchored Ras by combining attenuated total reflectance Fourier transform infrared spectroscopy, biomolecular simulations, and Förster resonance energy transfer experiments. By analyzing x-ray structural models and molecular-dynamics simulations, we propose a dimerization interface between α-helices 4 and 5 and the loop between β2 and β3. This seems to explain why the residues D47, E49, R135, R161, and R164 of this interface are influencing Ras signaling in cellular physiological experiments, although they are not positioned in the catalytic site. Dimerization could catalyze nanoclustering, which is well accepted for membrane-bound Ras. The interface could provide a new target for a seemingly novel type of small molecule interfering with signal transduction in oncogenic Ras mutants.     

BH3‐in‐groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes

Pro‐apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site‐specific disulfide crosslinking, compartment‐specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3‐in‐groove dimer interface on the MOM surface similar to that observed in crystals. However, after the α5 helix was released into the MOM, the remaining interface with α2, α3, and α4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel α9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3‐in‐groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, α9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by α9 insertion into the MOM and localization to the pore rim. Therefore, the BH3‐in‐groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by α9 dimerization at the rim.     

In-silico analysis of caspase-3 and -7 proteases from blood-parasitic Schistosoma species (Trematoda) and their human host

Proteolytic enzymes of the caspase family, which reside as latent precursors in most nucleated metazoan cells, are core effectors of apoptosis. Of them, the executioner caspases- 3 and -7 exist within the cytosol as inactive dimers and are activated by a process called dimerization. Caspase inhibition is looked upon as a promising approach for treating multiple diseases. Though caspases have been extensively studied in the human system, their role in eukaryotic pathogens and parasites of human hosts has not drawn enough attention. In protein sequence analysis, caspases of blood flukes (Schistosoma spp) were revealed to have a low sequence identity with their counterparts in human and other mammalian hosts, which encouraged us to analyse interacting domains that participate in dimerization of caspases in the parasite and to reveal differences, if any, between the host-parasite systems. Significant differences in the molecular surface arrangement of the dimer interfaces reveal that in schistosomal caspases only eight out of forty dimer conformations are similar to human caspase structures. Thus, the parasite-specific dimer conformations (that are different from caspases of the host) may emerge as potential drug targets of therapeutic value against schistosomal infections. Three important factors namely, the size of amino acids, secondary structures and geometrical arrangement of interacting domains influence the pattern of caspase dimer formation, which, in turn, is manifested in varied structural conformations of caspases in the parasite and its human hosts.     

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β₂-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β₂-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β₂-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.     

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

CLC-ec1 is a Cl⁻/H⁺ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4–5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.     

Functional role of dimerization of human peptidylarginine deiminase 4 (PAD4)

Peptidylarginine deiminase 4 (PAD4) is a homodimeric enzyme that catalyzes Ca²⁺-dependent protein citrullination, which results in the conversion of arginine to citrulline. This paper demonstrates the functional role of dimerization in the regulation of PAD4 activity. To address this question, we created a series of dimer interface mutants of PAD4. The residues Arg8, Tyr237, Asp273, Glu281, Tyr435, Arg544 and Asp547, which are located at the dimer interface, were mutated to disturb the dimer organization of PAD4. Sedimentation velocity experiments were performed to investigate the changes in the quaternary structures and the dissociation constants (K _d) between wild-type and mutant PAD4 monomers and dimers. The kinetic data indicated that disrupting the dimer interface of the enzyme decreases its enzymatic activity and calcium-binding cooperativity. The K _d values of some PAD4 mutants were much higher than that of the wild-type (WT) protein (0.45 µM) and were concomitant with lower k _cat values than that of WT (13.4 s⁻¹). The K _d values of the monomeric PAD4 mutants ranged from 16.8 to 45.6 µM, and the k _cat values of the monomeric mutants ranged from 3.3 to 7.3 s⁻¹. The k _cat values of these interface mutants decreased as the K _d values increased, which suggests that the dissociation of dimers to monomers considerably influences the activity of the enzyme. Although dissociation of the enzyme reduces the activity of the enzyme, monomeric PAD4 is still active but does not display cooperative calcium binding. The ionic interaction between Arg8 and Asp547 and the Tyr435-mediated hydrophobic interaction are determinants of PAD4 dimer formation.     

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β₂-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β₂-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β₂-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.     

Limiting an antimicrobial peptide to the lipid-water interface enhances its bacterial membrane selectivity: a case study of MSI-367

In a minimalist design approach, a synthetic peptide MSI-367 [(KFAKKFA)(3)-NH(2)] was designed and synthesized with the objective of generating cell-selective nonlytic peptides, which have a significant bearing on cell targeting. The peptide exhibited potent activity against both bacteria and fungi, but no toxicity to human cells at micromolar concentrations. Bacterial versus human cell membrane selectivity of the peptide was determined via membrane permeabilization assays. Circular dichroism investigations revealed the intrinsic helix propensity of the peptide, β-turn structure in aqueous buffer and extended and turn conformations upon binding to lipid vesicles. Differential scanning calorimetry experiments with 1,2-dipalmitoleoyl-sn-glycero-3-phosphatidylethanolamine bilayers indicated the induction of positive curvature strain and repression of the fluid lamellar to inverted hexagonal phase transition by MSI-367. Results of isothermal titration calorimetry (ITC) experiments suggested the possibility of formation of specific lipid-peptide complexes leading to aggregation. (2)H nuclear magnetic resonance (NMR) of deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) multilamellar vesicles confirmed the limited effect of the membrane-embedded peptide at the lipid-water interface. (31)P NMR data indicated changes in the lipid headgroup orientation of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine lipid bilayers upon peptide binding. Membrane-embedded and membrane-inserted states of the peptide were observed via sum frequency generation vibrational spectroscopy. Circular dichroism, ITC, and (31)P NMR data for Escherichia coli lipids agree with the hypothesis that strong electrostatic lipid-peptide interactions embrace the peptide at the lipid-water interface and provide the basis for bacterial cell selectivity.