Not Just an Oil Slick: How the Energetics of Protein-Membrane Interactions Impacts the Function and Organization of Transmembrane Proteins

The membrane environment, its composition, dynamics, and remodeling, have been shown to participate in the function and organization of a wide variety of transmembrane (TM) proteins, making it necessary to study the molecular mechanisms of such proteins in the context of their membrane settings. We review some recent conceptual advances enabling such studies, and corresponding computational models and tools designed to facilitate the concerted experimental and computational investigation of protein-membrane interactions. To connect productively with the high resolution achieved by cognate experimental approaches, the computational methods must offer quantitative data at an atomistically detailed level. We show how such a quantitative method illuminated the mechanistic importance of a structural characteristic of multihelical TM proteins, that is, the likely presence of adjacent polar and hydrophobic residues at the protein-membrane interface. Such adjacency can preclude the complete alleviation of the well-known hydrophobic mismatch between TM proteins and the surrounding membrane, giving rise to an energy cost of residual hydrophobic mismatch. The energy cost and biophysical formulation of hydrophobic mismatch and residual hydrophobic mismatch are reviewed in the context of their mechanistic role in the function of prototypical members of multihelical TM protein families: 1), LeuT, a bacterial homolog of mammalian neurotransmitter sodium symporters; and 2), rhodopsin and the β1- and β2-adrenergic receptors from the G-protein coupled receptor family. The type of computational analysis provided by these examples is poised to translate the rapidly growing structural data for the many TM protein families that are of great importance to cell function into ever more incisive insights into mechanisms driven by protein-ligand and protein-protein interactions in the membrane environment.     

Not Just Fat: The Structure and Function of the Lipid Droplet

Lipid droplets (LDs) are independent organelles that are composed of a lipid ester core and a surface phospholipid monolayer. Recent studies have revealed many new proteins, functions, and phenomena associated with LDs. In addition, a number of diseases related to LDs are beginning to be understood at the molecular level. It is now clear that LDs are not an inert store of excess lipids but are dynamically engaged in various cellular functions, some of which are not directly related to lipid metabolism. Compared to conventional membrane organelles, there are still many uncertainties concerning the molecular architecture of LDs and how each function is placed in a structural context. Recent findings and remaining questions are discussed.Lipid droplets (LDs) were recognized as a mere deposit of lipid esters for many years. Recently, LDs have been redefined as authentic organelles that are actively engaged in multiple functions, and these have been reviewed in many recent articles (Murphy 2001; Martin et al. 2006; Ducharme et al. 2008; Fujimoto et al. 2008; Goodman 2008; Olofsson et al. 2008; Thiele et al. 2008; Murphy et al. 2009; Walther et al. 2009; Beller et al. 2010). The name, lipid droplets, implies the opposite: a static and inert nature. Alternative names have been proposed, but lipid droplet is now a generally agreed nomenclature (Martin et al. 2006).LDs are intimately related to many aspects of lipid metabolism. The lipid storage function of LDs is most conspicuous in white adipocytes, which have a gigantic unilocular LD. The regulation of the process of lipid storage and utilization has been the focus of many studies because of the prevalence of obesity, type II diabetes, and metabolic syndrome in the modern world.Besides the canonical lipid-related function, various new functions have been ascribed to LDs more recently, including some that do not appear to be directly related to lipid metabolism. However, both the canonical and noncanonical functions of LDs, are rooted in the unique architecture of LDs. In contrast to other organelles that have aqueous content within a phospholipid bilayer membrane, the basic structure of LDs is thought to be a mass of lipid esters covered by a phospholipid monolayer (Murphy et al. 1999; Tauchi-Sato et al. 2002). In this article, we first review what is known about LD structure and then discuss various functions. We review this information by considering the extent to which the functions can be understood based on the current structural paradigm.     

Influence of Transmembrane Helix Mutations on Cytochrome P450-Membrane Interactions and Function

Human cytochrome P450 (CYP) enzymes play an important role in the metabolism of drugs, steroids, fatty acids, and xenobiotics. Microsomal CYPs are anchored in the endoplasmic reticulum membrane by an N-terminal transmembrane (TM) helix that is connected to the globular catalytic domain by a flexible linker sequence. However, the structural and functional importance of the TM-helix is unclear because it has been shown that CYPs can still associate with the membrane and have enzymatic activity in reconstituted systems after truncation or modification of the N-terminal sequence. Here, we investigated the effect of mutations in the N-terminal TM-helix residues of two human steroidogenic enzymes, CYP 17A1 and CYP 19A1, that are major drug targets for cancer therapy. These mutations were originally introduced to increase the expression of the proteins in Escherichia coli. To investigate the effect of the mutations on protein-membrane interactions and function, we carried out coarse-grained and all-atom molecular dynamics simulations of the CYPs in a phospholipid bilayer. We confirmed the orientations of the globular domain in the membrane observed in the simulations by linear dichroism measurements in a Nanodisc. Whereas the behavior of CYP 19A1 was rather insensitive to truncation of the TM-helix, mutations in the TM-helix of CYP 17A1, especially W2A and E3L, led to a gradual drifting of the TM-helix out of the hydrophobic core of the membrane. This instability of the TM-helix could affect interactions with the allosteric redox partner, cytochrome b5, required for CYP 17A1’s lyase activity. Furthermore, the simulations showed that the mutant TM-helix influenced the membrane interactions of the CYP 17A1 globular domain. In some simulations, the mutated TM-helix obstructed the substrate access tunnel from the membrane to the CYP active site, indicating a possible effect on enzyme function.     

Major mRNP Proteins in the Structural Organization and Function of mRNA in Eukaryotic Cells

The properties of two universal major proteins of cytoplasmic mRNP, p50 and the poly(A)-binding protein (PABP), are summarized. Their roles in formation of polyribosomal and free inactive mRNP are considered, with the focus on the authors' studies of p50. The parts these mRNP proteins play in translation regulation, stability, and localization of mRNA are described, and the possible mechanisms of their function are discussed.     

Evidence for New Homotypic and Heterotypic Interactions between Transmembrane Helices of Proteins Involved in Receptor Tyrosine Kinase and Neuropilin Signaling

Signaling in eukaryotic cells frequently relies on dynamic interactions of single-pass membrane receptors involving their transmembrane (TM) domains. To search for new such interactions, we have developed a bacterial two-hybrid system to screen for both homotypic and heterotypic interactions between TM helices. We have explored the dimerization of TM domains from 16 proteins involved in both receptor tyrosine kinase and neuropilin signaling. This study has revealed several new interactions. We found that the TM domain of Mucin-4, a putative intramembrane ligand for erbB2, dimerizes not only with erbB2 but also with all four members of the erbB family. In the Neuropilin/Plexin family of receptors, we showed that the TM domains of Neuropilins 1 and 2 dimerize with themselves and also with Plexin-A1, Plexin-B1, and L1CAM, but we were unable to observe interactions with several other TM domains notably those of members of the VEGF receptor family. The potentially important Neuropilin 1/Plexin-A1 interaction was confirmed using a surface plasmon resonance assay. This work shows that TM domain interactions can be highly specific. Exploring further the propensities of TM helix–helix association in cell membrane should have important practical implications related to our understanding of the structure–function of bitopic proteins' assembly and subsequent function, especially in the regulation of signal transduction.     

Impacts of Sediments on Coral Energetics: Partitioning the Effects of Turbidity and Settling Particles

Sediment loads have long been known to be deleterious to corals, but the effects of turbidity and settling particles have not previously been partitioned. This study provides a novel approach using inert silicon carbide powder to partition and quantify the mechanical effects of sediment settling versus reduced light under a chronically high sedimentary regime on two turbid water corals commonly found in Singapore (Galaxea fascicularis and Goniopora somaliensis). Coral fragments were evenly distributed among three treatments: an open control (30% ambient PAR), a shaded control (15% ambient PAR) and sediment treatment (15% ambient PAR; 26.4 mg cm⁻² day⁻¹). The rate of photosynthesis and respiration, and the dark-adapted quantum yield were measured once a week for four weeks. By week four, the photosynthesis to respiration ratio (P/R ratio) and the photosynthetic yield (F_v/F_m) had fallen by 14% and 3–17% respectively in the shaded control, contrasting with corals exposed to sediments whose P/R ratio and yield had declined by 21% and 18–34% respectively. The differences in rates between the shaded control and the sediment treatment were attributed to the mechanical effects of sediment deposition. The physiological response to sediment stress differed between species with G. fascicularis experiencing a greater decline in the net photosynthetic yield (13%) than G. somaliensis (9.5%), but a smaller increase in the respiration rates (G. fascicularis = 9.9%, G. somaliensis = 14.2%). These different physiological responses were attributed, in part, to coral morphology and highlighted key physiological processes that drive species distribution along high to low turbidity and depositional gradients.     

Control and Regulation of Mitochondrial Energetics in an Integrated Model of Cardiomyocyte Function

Understanding the regulation and control of complex networks of reactions requires analytical tools that take into account the interactions between individual network components controlling global network function. Here, we apply a generalized matrix method of control analysis to calculate flux and concentration control coefficients, as well as response coefficients, in an integrated model of excitation-contraction (EC) coupling and mitochondrial energetics (ECME model) in the cardiac ventricular myocyte. Control and regulation of oxygen consumption (V_O2) was first assessed in a mitochondrion model, and then in the integrated cardiac myocyte model under resting and working conditions. The results demonstrate that in the ECME model, control of respiration is distributed among cytoplasmic ATPases and mitochondrial processes. The magnitude of control by cytoplasmic ATPases increases under working conditions. The model prediction that the respiratory chain exerts strong positive control on V_O2 (control coefficient 0.89) was corroborated experimentally in cardiac trabeculae utilizing the inhibitor titration method. In the model, mitochondrial respiration displayed the highest response coefficients with respect to the concentration of cytoplasmic ATP. This was due to the high elasticity of ANT flux toward ATP in the cytoplasm. The analysis reveals the complex interdependence of sarcolemmal, cytoplasmic, and mitochondrial processes that contribute to the control of energy supply and demand in the heart. Moreover, by visualizing the structure of control of the metabolic network of the myocyte, we provide support for the emerging concept of control by diffuse loops, in which action on the network (e.g., by a pharmacological agent) may bring about changes in processes without obvious direct mechanistic links between them.     

Interactions between an alpha-helix and a beta-sheet. Energetics of alpha/beta packing in proteins

Conformational energy computations have been carried out to determine the favorable ways of packing a right-handed alpha-helix on a right-twisted antiparallel or parallel beta-sheet. Co-ordinate transformations have been developed to relate the position and orientation of the alpha-helix to the beta-sheet. The packing was investigated for a CH3CO-(L-Ala)16-NHCH3 alpha-helix interacting with five-stranded beta-sheets composed of CH3CO-(L-Val)6-NHCH3 chains. All internal and external variables for both the alpha-helix and the beta-sheet were allowed to change during energy minimization. Four distinct classes of low-energy packing arrangements were found for the alpha-helix interacting with both the parallel and the anti-parallel beta-sheet. The classes differ in the orientation of the axis of the alpha-helix relative to the direction of the strands of the right-twisted beta-sheet. In the class with the most favorable arrangement, the alpha-helix is oriented along the strands of the beta-sheet, as a result of attractive non-bonded side-chain-side-chain interactions along the entire length of the alpha-helix. A class with nearly perpendicular orientation of the helix axis to the strands is also of low energy, because it allows similarly extensive attractive interactions. In the other two classes, the helix is oriented diagonally relative to the strands of the beta-sheet. In one of them, it interacts with the convex surface near the middle of the saddle-shaped twisted beta-sheet. In the other, it is oriented along the concave diagonal of the beta-sheet and, therefore, it interacts only with the corner regions of the sheet, so that this packing is energetically less favorable. The packing arrangements involving an antiparallel and a parallel beta-sheet are generally similar, although the antiparallel beta-sheet has been found to be more flexible. The major features of 163 observed alpha/beta packing arrangements in 37 proteins are accounted for in terms of the computed structural preferences. The energetically most favored packing arrangement is similar to the right-handed beta alpha beta crossover structure that is observed in proteins; thus, the preference for this connectivity arises in large measure from this energetically favorable interaction.     

Coordination of an Array of Signaling Proteins through Homo- and Heteromeric Interactions Between PDZ Domains and Target Proteins

The rapid activation and feedback regulation of many G protein signaling cascades raises the possibility that the critical signaling proteins may be tightly coupled. Previous studies show that the PDZ domain containing protein INAD, which functions in Drosophila vision, coordinates a signaling complex by binding directly to the light-sensitive ion channel, TRP, and to phospholipase C (PLC). The INAD signaling complex also includes rhodopsin, protein kinase C (PKC), and calmodulin, though it is not known whether these proteins bind to INAD. In the current work, we show that rhodopsin, calmodulin, and PKC associate with the signaling complex by direct binding to INAD. We also found that a second ion channel, TRPL, bound to INAD. Thus, most of the proteins involved directly in phototransduction appear to bind to INAD. Furthermore, we found that INAD formed homopolymers and the homomultimerization occurred through two PDZ domains. Thus, we propose that the INAD supramolecular complex is a higher order signaling web consisting of an extended network of INAD molecules through which a G protein–coupled cascade is tethered.     

Analysis of the Contribution of Wind Drift Factor to Oil Slick Movement under Strong Tidal Condition: Hebei Spirit Oil Spill Case

The purpose of this study is to investigate the effects of the wind drift factor under strong tidal conditions in the western coastal area of Korea on the movement of oil slicks caused by the Hebei Spirit oil spill accident in 2007. The movement of oil slicks was computed using a simple simulation model based on the empirical formula as a function of surface current, wind speed, and the wind drift factor. For the simulation, the Environmental Fluid Dynamics Code (EFDC) model and Automatic Weather System (AWS) were used to generate tidal and wind fields respectively. Simulation results were then compared with 5 sets of spaceborne optical and synthetic aperture radar (SAR) data. From the present study, it was found that highest matching rate between the simulation results and satellite imagery was obtained with different values of the wind drift factor, and to first order, this factor was linearly proportional to the wind speed. Based on the results, a new modified empirical formula was proposed for forecasting the movement of oil slicks on the coastal area.     

Not Just an empty cavity: the inter-rhabdomeral space in the Jamaican cavefly Neoditomyia farri (Diptera, Mycetophilidae)

Contrary to most other Diptera, the inter-rhabdomeral spaces of the retina of the Jamaican cavefly Neoditomya farri are filled neither by extracellular matrix nor dense cytoplasmic material. Instead, a foamy organization of loose vacuoles, measuring approximately 0.7 microm in diameter, appears to keep the rhabdomeres of retinula cells 7 and 8 in place. The vacuoles are bounded by membranes and traces of actin, determined immunocytochemically, are present. The origin of the vacuoles is unclear, but evidence in support of a retinula cell rather than cone cell origin is advanced.     

Polarized Organization of the Cytoskeleton: Regulation by Cell Polarity Proteins

Polarity is one of the fundamental properties displayed by living organisms. In metazoans, cell polarity governs developmental processes and plays an essential role during maintenance of forms of tissues as well as their functions. The mechanisms of establishment and maintenance of cell polarity have been investigated extensively in the last two decades. This has resulted in identification of “core cell polarity modules” that control anterior–posterior, front–rear and apical–basal polarity across various cell types. Here, we review how these polarity modules interact closely with the cytoskeleton during establishment and maintenance of cytoskeletal polarity. We further suggest that reciprocal interactions between cell polarity modules and the cytoskeleton consolidate the initial weaker polarity, arising from an external cue, into a committed polarized system.     

Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins

The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXØ, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXØ motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXØ motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXØ motifs interact with the μ1 and μ2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the β2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXØ-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.     

Predicting Interaction Sites from the Energetics of Isolated Proteins: A New Approach to Epitope Mapping

An increasing number of functional studies of proteins have shown that sequence and structural similarities alone may not be sufficient for reliable prediction of their interaction properties. This is particularly true for proteins recognizing specific antibodies, where the prediction of antibody-binding sites, called epitopes, has proven challenging. The antibody-binding properties of an antigen depend on its structure and related dynamics. Aiming to predict the antibody-binding regions of a protein, we investigate a new approach based on the integrated analysis of the dynamical and energetic properties of antigens, to identify nonoptimized, low-intensity energetic interaction networks in the protein structure isolated in solution. The method is based on the idea that recognition sites may correspond to localized regions with low-intensity energetic couplings with the rest of the protein, which allows them to undergo conformational changes, to be recognized by a binding partner, and to tolerate mutations with minimal energetic expense. Upon analyzing the results on isolated proteins and benchmarking against antibody complexes, it is found that the method successfully identifies binding sites located on the protein surface that are accessible to putative binding partners. The combination of dynamics and energetics can thus discriminate between epitopes and other substructures based only on physical properties. We discuss implications for vaccine design.     

Analysis of the DNA-Binding Profile and Function of TALE Homeoproteins Reveals Their Specialization and Specific Interactions with Hox Genes/Proteins

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Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor

Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 Å crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120^GAP·H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation.