Membrane protein insertase YidC in bacteria and archaea

The insertion of proteins into the prokaryotic plasma membrane is catalyzed by translocases and insertases. On one hand, the Sec translocase operates as a transmembrane channel that can open laterally to first bind and then release the hydrophobic segments of a substrate protein into the lipid bilayer. On the other hand, YidC insertases interact with their substrates in a groove‐like structure at an amphiphilic protein–lipid interface thus allowing the transmembrane segments of the substrate to slide into the lipid bilayer. The recently published high‐resolution structures of YidC provide new mechanistic insights of how transmembrane proteins achieve the transition from an aqueous environment in the cytoplasm to the hydrophobic lipid bilayer environment of the membrane.     

The role of lipids in the biogenesis of integral membrane proteins

Most integral membrane proteins are cotranslationally inserted into the lipid bilayer. In prokaryotes, membrane insertion of the nascent chain takes place at the plasma membrane, whereas in eukaryotes insertion takes place into the endoplasmatic reticulum. In both kingdoms of life, however, the same membrane that acquaints the newly born membrane protein also synthesizes the bilayer lipids and thus ensures the balanced growth of the membrane as a whole. Recent evidence indicates that the lipid composition of the host membrane can determine the fate of the newborn membrane protein, as it can affect (1) the efficiency of translocation, (2) the topology of the resulting membrane protein, (3) its stability, (4) its assembly into oligomeric complexes, (5) its transport and sorting along the secretory pathway, and (6) its enzymatic activity. The lipid composition of the membrane thus can affect the biogenesis and function of integral membrane proteins at multiple steps along its biogenetic pathway. While understanding this interdependence between bilayer lipids and protein biogenesis is interesting in its own right, careful consideration of a potential host’s membrane lipid composition may also allow optimization of the yield and activity of membrane proteins that are expressed in a heterologous organism. Here, we review and discuss some examples that illustrate the interdependence between bilayer lipids and the biogenesis of integral membrane proteins.     

Membrane biology: fission behind BARs

Membrane bending is accomplished in part by amphipathic helix insertion into the bilayer and the assembly of BAR domain scaffolds preparing the membrane for fission. Two recent studies highlight the roles of amphipathic helices and BAR scaffolds in membrane fission and establish the structural basis of membrane bending by the N-BAR protein endophilin.     

Fishing for biomarkers with antigen mimics

The transmembrane domains in a membrane protein must be recognized and correctly oriented before their insertion into the lipid bilayer. Devaraneni et?al. (2011) generate snapshots at different stages of membrane protein biogenesis, revealing a dynamic set of steps that imply an unexpectedly flexible membrane insertion machinery.     

Membrane insertion of Escherichia coli alpha-hemolysin is independent from membrane lysis

Escherichia coli alpha-hemolysin (HlyA) is a protein exotoxin that binds and lyses eukaryotic cell and model membranes in the presence of calcium. Previous studies have been able to distinguish between reversible toxin binding to the membrane and irreversible insertion into the lipid matrix. Membrane lysis occurs as the combined effect of protein insertion plus a transient perturbation of the membrane bilayer structure. In the past, insertion and bilayer perturbation have not been experimentally dissected. This has now been achieved by studying HlyA penetration into lipid monolayers at the air-water interface, in which three-dimensional effects (of the kind required to break down the bilayer permeability barrier) cannot occur. The study of native HlyA, together with the nonlytic precursor pro-HlyA, and of different mutants demonstrates that although some nonlytic variants (e.g. pro-HlyA) exhibit very low levels of insertion, others (e.g. the nonlytic mutant HlyA H859N) insert even more strongly than the lytic wild type. These results show that insertion does not necessarily lead to membrane lysis, i.e. that insertion and lysis are not "coupled" phenomena. Millimolar levels of Ca(2+), which are essential for the lytic activity, cause an extra degree of insertion but only in the case of the lytic forms of HlyA.     

Determining Peptide Partitioning Properties via Computer Simulation

The transfer of polypeptide segments into lipid bilayers to form transmembrane helices represents the crucial first step in cellular membrane protein folding and assembly. This process is driven by complex and poorly understood atomic interactions of peptides with the lipid bilayer environment. The lack of suitable experimental techniques that can resolve these processes both at atomic resolution and nanosecond timescales has spurred the development of computational techniques. In this review, we summarize the significant progress achieved in the last few years in elucidating the partitioning of peptides into lipid bilayer membranes using atomic detail molecular dynamics simulations. Indeed, partitioning simulations can now provide a wealth of structural and dynamic information. Furthermore, we show that peptide-induced bilayer distortions, insertion pathways, transfer free energies, and kinetic insertion barriers are now accurate enough to complement experiments. Further advances in simulation methods and force field parameter accuracy promise to turn molecular dynamics simulations into a powerful tool for investigating a wide range of membrane active peptide phenomena.     

The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition

The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane alpha-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-l-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane alpha-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces.     

Secondary and tertiary structure formation of the beta-barrel membrane protein OmpA is synchronized and depends on membrane thickness

The mechanism of membrane insertion and folding of a beta-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example. OmpA forms an eight-stranded beta-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli. OmpA folds spontaneously from a urea-denatured state into lipid bilayers of small unilamellar vesicles. We have used fluorescence spectroscopy, circular dichroism spectroscopy, and gel electrophoresis to investigate basic mechanistic principles of structure formation in OmpA. Folding kinetics followed a second-order rate law and is strongly depended on the hydrophobic thickness of the lipid bilayer. When OmpA was refolded into model membranes of dilaurylphosphatidylcholine, fluorescence kinetics were characterized by a rate constant that was about fivefold higher than the rate constants of formation of secondary and tertiary structure, which were determined by circular dichroism spectroscopy and gel electrophoresis, respectively. The formation of beta-sheet secondary structure and closure of the beta-barrel of OmpA were correlated with the same rate constant and coupled to the insertion of the protein into the lipid bilayer. OmpA, and presumably other beta-barrel membrane proteins therefore do not follow a mechanism according to the two-stage model that has been proposed for the folding of alpha-helical bundle membrane proteins. These different folding mechanisms are likely a consequence of the very different intramolecular hydrogen bonding and hydrophobicity patterns in these two classes of membrane proteins.     

Novel preparation of functional Sindbis virosomes

Lipid and protein factors important for the preparation and stability of reconstituted membranes prepared by the insertion of detergent-solubilized Sindbis virus glycoproteins into preformed lipid vesicles have been defined. It was found that both the state of aggregation of the membrane protein and the phase of the lipid are critical for the insertion of proteins into preformed lipid vesicles. The membranes prepared with the insertion technique were characterized in terms of residual detergent, protein orientation, and whether or not they were sealed. Binding and fusion experiments have been carried out with the insertion membranes and virus. It was found that BHK-21 cells at 4 degrees C bound one-fifth to one-tenth the number of insertion membranes as intact virus, and binding was saturable in both cases. Variation of the lipid/protein ratio did not result in significant differences in binding. The insertion membranes were found to fuse to a model lipid bilayer at least as well as the virus. These results are discussed in terms of structural factors important for the biological functionalities of the viral spike glycoproteins.     

Exploring peptide-membrane interactions with coarse-grained MD simulations

The interaction of α-helical peptides with lipid bilayers is central to our understanding of the physicochemical principles of biological membrane organization and stability. Mutations that alter the position or orientation of an α-helix within a membrane, or that change the probability that the α-helix will insert into the membrane, can alter a range of membrane protein functions. We describe a comparative coarse-grained molecular dynamics simulation methodology, based on self-assembly of a lipid bilayer in the presence of an α-helical peptide, which allows us to model membrane transmembrane helix insertion. We validate this methodology against available experimental data for synthetic model peptides (WALP23 and LS3). Simulation-based estimates of apparent free energies of insertion into a bilayer of cystic fibrosis transmembrane regulator-derived helices correlate well with published data for translocon-mediated insertion. Comparison of values of the apparent free energy of insertion from self-assembly simulations with those from coarse-grained molecular dynamics potentials of mean force for model peptides, and with translocon-mediated insertion of cystic fibrosis transmembrane regulator-derived peptides suggests a nonequilibrium model of helix insertion into bilayers.     

Kinetic study of folding and misfolding of diacylglycerol kinase in model membranes

Despite the relevance of membrane protein misfolding to a number of common diseases, our understanding of the folding and misfolding of membrane proteins lags well behind soluble proteins. Here, the overall kinetics of membrane insertion and folding of the homotrimeric integral membrane protein diacylglycerol kinase (DAGK) is addressed. DAGK was purified into lipid/detergent-free urea and guanidinium solutions and subjected to general structural characterization. In urea, the enzyme was observed to be monomeric but maintained considerable tertiary structure. In guanidinium, it was also monomeric but exhibited much less tertiary structure. Aliquots of these DAGK stock solutions were diluted 200-fold into lipid vesicles or into detergent/lipid mixed micelles, and the rates and efficiencies of folding/insertion were monitored. Reactions were also carried out in which micellar DAGK solutions were diluted into vesicular solutions. Productive insertion of DAGK from denaturant solutions into mixed micelles occurred much more rapidly than into lipid vesicles, suggesting that bilayer transversal represents the rate-limiting step for DAGK assembly in vesicles. The efficiency of productive folding/insertion into vesicles was highest in reactions initiated with micellar DAGK stock solutions (where DAGK maintains a nativelike fold and oligomeric state) and lowest in reactions starting with guanidinium stocks (where DAGK is an unfolded monomer). Moreover, the final ratio of irreversibly misfolded DAGK to reversibly misfolded enzyme was highest following reactions initiated with guanidinium stock solutions and lowest when micellar stocks were used. Finally, it was also observed that very low concentrations of detergents were able to both enhance the bilayer insertion rate and suppress misfolding.     

YidC,a newly defined evolutionarily conserved protein,mediates membrane protein assembly in bacteria

Membranes contain proteins that catalyze a variety of reactions, which lead to the selective permeability of the membrane. For membrane proteins to function as receptors, transporters, channels, and ATPases, they must be targeted to their correct membrane and inserted into the lipid bilayer. Recently, a new membrane component called YidC was discovered that mediates the insertion of proteins into membranes in bacteria. YidC homologs also exist in mitochondria and chloroplasts. Depletion of YidC from the cell interferes with the insertion of membrane proteins that insert both dependent and independent of the SecYEG/SecDFYajC machinery. YidC directly interacts with membrane proteins during the membrane protein insertion process and assists in the folding of the hydrophobic regions into the membrane bilayer. The chloroplast and bacterial YidC homologs are truly functional homologs because the chloroplast homolog Alb3 functionally complements the bacterial YidC depletion strain. The role of YidC in the membrane insertion pathway will be reviewed.     

Membrane protein biosynthesis - all sewn up?

The biosynthesis of integral membrane proteins requires regions of nascent polypeptide to be inserted into, and assembled within, a lipid bilayer. Membrane protein integration at the endoplasmic reticulum (ER) has been shown to involve a defined set of protein components. In addition, thephospholipid bilayer may also make a specific contribution to a functional ER integration site. This review focuses on recent studies of membrane protein insertion and subsequent maturation events, both of which are prerequisites for the synthesis of functional integral membrane proteins.     

Folding and assembly of beta-barrel membrane proteins

Beta-barrel membrane proteins occur in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The membrane-spanning sequences of beta-barrel membrane proteins are less hydrophobic than those of alpha-helical membrane proteins, which is probably the main reason why completely different folding and membrane assembly pathways have evolved for these two classes of membrane proteins. Some beta-barrel membrane proteins can be spontaneously refolded into lipid bilayer model membranes in vitro. They may also have this ability in vivo although lipid and protein chaperones likely assist with their assembly in appropriate target membranes. This review summarizes recent work on the thermodynamic stability and the mechanism of membrane insertion of beta-barrel membrane proteins in lipid model and biological membranes. How lipid compositions affect folding and assembly of beta-barrel membrane proteins is also reviewed. The stability of these proteins in membranes is not as large as previously thought (<10 kcal/mol) and is modulated by elastic forces of the lipid bilayer. Detailed kinetic studies indicate that beta-barrel membrane proteins fold in distinct steps with several intermediates that can be characterized in vitro. Formation of the barrel is synchronized with membrane insertion and all beta-hairpins insert simultaneously in a concerted pathway.     

Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.     

In vitro folding of KvAP, a voltage-gated K+ channel

In this contribution, we report in vitro folding of the archaebacterial voltage-gated K(+) channel, K(v)AP. We show that in vitro folding of the K(v)AP channel from the extensively unfolded state requires lipid vesicles and that the refolded channel is biochemically and functionally similar to the native channel. The in vitro folding process is slow at room temperature, and the folding yield depends on the composition of the lipid bilayer. The major factor influencing refolding is temperature, and almost quantitative refolding of the K(v)AP channel is observed at 80 °C. To differentiate between insertion into the bilayer and folding within the bilayer, we developed a cysteine protection assay. Using this assay, we demonstrate that insertion of the unfolded protein into the bilayer is relatively fast at room temperature and independent of lipid composition, suggesting that temperature and bilayer composition influence folding within the bilayer. Further, we demonstrate that in vitro folding provides an effective method for obtaining high yields of the native channel. Our studies suggest that the K(v)AP channel provides a good model system for investigating the folding of a multidomain integral membrane protein.     

The two membrane segments of leader peptidase partition one by one into the lipid bilayer via a Sec/YidC interface

We have addressed the mechanism of insertion of both transmembrane segments (TMs) of leader peptidase, a double-spanning protein, into the Escherichia coli inner membrane. Using photo-crosslinking, the first TM (H1) was shown to insert at a Sec-translocon/YidC interface in a fixed orientation. H1 lost its contacts with the Sec-translocon and gained access to lipids near YidC soon after complete exposure outside the ribosome. Following lipid integration, it moved away from the Sec/YidC insertion site. The second TM (H2) inserted and interacted with SecY and YidC in a similar transient fashion. The data are consistent with a linear integration model in which the TMs of polytopic inner membrane proteins move one by one from a Sec/YidC insertion site into the lipid bilayer. We propose that YidC assists the lipid partitioning of single TMs.     

Folding is not required for bilayer insertion: replica exchange simulations of an alpha-helical peptide with an explicit lipid bilayer

We implement the replica exchange molecular dynamics algorithm to study the interactions of a model peptide (WALP-16) with an explicitly represented DPPC membrane bilayer. We observe the spontaneous, unbiased insertion of WALP-16 into the DPPC bilayer and its folding into an alpha-helix with a transbilayer orientation. The free energy surface suggests that the insertion of the peptide into the DPPC bilayer precedes secondary structure formation. Although the peptide has some propensity to form a partially helical structure in the interfacial region of the DPPC/water system, this state is not a productive intermediate but rather an off-pathway trap for WALP-16 insertion. Equilibrium simulations show that the observed insertion/folding pathway mirrors the potential of mean force (PMF). Calculation of the enthalpic and entropic contributions to this PMF show that the surface bound conformation of WALP-16 is significantly lower in energy than other conformations, and that the insertion of WALP-16 into the bilayer without regular secondary structure is enthalpically unfavorable by 5-10 kcal/mol/residue. The observed insertion/folding pathway disagrees with the dominant conceptual model, which is that a surface-bound helix is an obligatory intermediate for the insertion of alpha-helical peptides into lipid bilayers. In our simulations, the observed insertion/folding pathway is favored because of a large (>100 kcal/mol) increase in system entropy that occurs when the unstructured WALP-16 peptide enters the lipid bilayer interior. The insertion/folding pathway that is lowest in free energy depends sensitively on the near cancellation of large enthalpic and entropic terms. This suggests the possibility that intrinsic membrane peptides may have a diversity of insertion/folding behaviors depending on the exact system of peptide and lipid under consideration.     

Electrostatic free energy and spontaneous curvature of spherical charged layered membrane

A biophysical model for the equilibrium curvature of a composite membrane element is derived taking into account the mechanical bilayer properties and the adjacent charged protein layers. The minimum of the total free energy density with respect to the curvature of such a membrane curved was estimated from the sum of the electrostatic free energy density of the charges of the membrane and the elastic surface energy density due to bending the lipid bilayer membrane. It was shown that the equilibrium curvature, i.e. the spontaneous curvature, of such a charged composite sandwich-like membrane depends inversely on the bending stiffness of the lipid membrane itself and directly on the charge amount inside and outside the membrane to the second power. Furthermore the geometric and electrostatic structure of the protein layers and the physico-chemical environment conditions are involved. Corresponding to the model developed a "standard RBC" membrane element has a negative spontaneous curvature, accounting for a discocyte RBC shape. The shape change from a discocyte to a more stomatocytic shape (increase in the negative spontaneous curvature) after reducing the charges in the glycocalyx is also explained within this model.     

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies.