Structural requirements for a primitive adaptor molecule

Adaptor properties of linear hairpin helices have been examined. The analysis suggests that neither right nor left handed hairpin helices can simultaneously read a comma free messenger and align aminoacyl residues for peptide condensation. Comparison of these studies with the model of the present day peptidyl transfer intermediate suggests that the "L" shaped folding of the present day tRNAs may be a prerequisite for adaptor function. Therefore, the three-dimensional organization of the ancestral adaptor molecule must have had structural features similar to its present day counterpart.     

Structural elements and organization of the ancestral translational machinery

The molecular mechanisms underlying the primitive translational apparatus have been studied in light of present day protein biosynthesis. Using the structural information available from the contemporary system as a key to its function, both the structural necessities for an early adaptor and the multipoint recognition properties of such adaptors have been investigated. This was done by first critically examining the potential feasibility of right- and left-handed hairpin adaptor models. Second, a molecular model of the contemporary transpeptidation complex has been constructed in order to ascertain the structural requirements of the adaptor molecule needed for peptidyl transfer. Third, a model of the tRNA^Tyr-tyrosyl tRNA synthetase complex including the positioning of the disordered region is proposed. This model is used to illustrate those required recognition properties of aminoacyl synthetase which lead to a perspective on the structure of the ancestor synthetase.     

Free energy of imperfect nucleic acid helices. II. Small hairpin loops

Physical studies of enzymically synthesized oligonucleotides of defined sequence are used to evaluate quantitatively the stability of small RNA hairpin loops and helices. The series (Ap)₄G(pC) _N(pU)₄, N = 4, 5 or 6, exists as monomolecular hairpin helices when N ≥ 5, and as imperfect dimer helices when N ≤ 4. In this size range, hairpin loops become more favorable (less destabilizing thermodynamically) as they increase in size from 3 to 4 to 5 unbonded nucleotides. Very small hairpin loops are particularly destabilizing; molecules whose base sequence would imply a hairpin loop of three nucleotides will generally exist with a loop of five, including a broken terminal base pair.Thermodynamic parameters for base pair and loop formation are calculated by a method which makes unnecessary the use of measured enthalpies of polynucleotide melting. Literature data on oligonucleotide double helices yield estimates of the free energy contribution from each of the six types of stacking interactions between three possible neighboring base pairs. The advantage of this approach is that the properties of oligonucleotides are used in predicting the stability of small RNA helices, avoiding the long extrapolation from the properties of high polymers.We provide Tables of temperature-dependent free energies that allow one to predict the stability and thermal transition temperature of many simple RNA secondary structures (applicable to ~1 m-Na⁺ concentration). As an example, we apply the rules to an isolated fragment of tRNA^Ser (yeast) (Coutts, 1971), whose properties were not used in calculating the free-energy parameters. The experimental melting temperature of 88 °C is predicted with an error margin of 5 deg. C.     

The spontaneous insertion of proteins into and across membranes: The helical hairpin hypothesis

We propose that the initial event in the secretion of proteins across membranes and their insertion into membranes is the spontaneous penetration of the hydrophobic portion of the bilayer by a helical hairpin. Energetic considerations of polypeptide structures in a nonpolar, lipid environment compared with an aqueous environment suggest that only α and 3₁₀ helices will be observed in the hydrophobic interior of membranes. Insertion of a polypeptide is accomplished by a hairpin structure composed of two helices, which will partition into membranes if the free energy arising from burying hydrophobic helical surfaces exceeds the free energy “cost” of burying potentially charged and hydrogen-bonding groups. We suggest, for example, that the hydrophobic leader peptide found in secreted proteins and in many membrane proteins forms one of these helices and is oriented in the membrane with its N terminus inside. In secreted proteins, the leader functions by pulling polar portions of a protein into the membrane as the second helix of the hairpin. The occurrence of all categories of membrane proteins can be rationalized by the hydrophobic or hydrophilic character of the two helices of the inserted hairpin and, for some integral membrane proteins, by events in which a single terminal helix is inserted. We propose that, because of the distribution of polar and nonpolar sequences in the polypeptide sequence, secretion and the insertion of membrane proteins are spontaneous processes that do not require the participation of additional specific membrane receptors or transport proteins.     

Terminal sidechain packing of a designed β‐hairpin influences conformation and stability

While end capping in α‐helices is well understood, the concept of capping a β‐hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a β‐hairpin. While cross‐strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed β‐hairpin a conservative, hydrophobic, V → I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H‐bonded site and the sidechain at the C‐terminal non‐H‐bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C‐terminal site, for example an E → A mutation (ΔΔG_U = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 557–564, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Binding of Bis-linked Netropsin Derivatives in the Parallel-Stranded Hairpin Form to DNA

Abstract

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5′-CCTATATCC-3′ in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis -diammine Pt(II)- bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5′-CCTATATCC-3′ (I), 5′-CCTTAATCC-3′ (II), 5′-CCTTATTCC-3′ (III), 5′-CCTTTTTCC-3′ (IV) and 5′-CCAATTTCC-3′ (V) decreases in the order I = II > III > IV> V. The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.     

Loopstructures in synthetic oligonucleotides. Hairpin stability and structure studied as a function of loop elongation

The formation of hairpin structures in the homologous, (partly) self-complementary DNA fragments d(ATCCTAT_nTAGGAT), n = 0–7, was studied by means of nuclear magnetic resonance, T-jump and ultra-violet techniques. It is shown that all compounds in the series may adopt hairpin-like conformations, albeit for n < 3 this only occurs to a significant amount at relatively low concentrations (∼ 10μM). For the present series of oligonucleotides, hairpin formation is accompanied by an apparent loop enthalpy significantly different from zero. The stability of the DNA hairpins turns out to be at its maximum for loop lengths of four or five residues, whereas earlier experiments (Tinocoet al., 1973) indicated that loop lengths of six to seven residues are most favourable for RNA hairpins. This is explained by considering the difference in geometry of A-RNA and B-DNA helices.     

Importance of the adaptor (membrane fusion) protein hairpin domain for the functionality of multidrug efflux pumps

Drug efflux pumps of Gram-negative bacteria are tripartite export machineries located in the bacterial envelopes contributing to multidrug resistance. Protein structures of all three components have been determined, but the exact interaction sites are still unknown. We could confirm that the hybrid system composed of Pseudomonas aeruginosa channel tunnel OprM and the Escherichia coli inner membrane complex, formed by adaptor protein (membrane fusion protein) AcrA and transporter AcrB of the resistance nodulation cell division (RND) family, is not functional. However, cross-linking experiments show that the hybrid exporter assembles. Exchange of the hairpin domain of AcrA with the corresponding hairpin from adaptor protein MexA of P. aeruginosa restored the functionality. This shows the importance of the MexA hairpin domain for the functional interaction with the OprM channel tunnel. On the basis of these results, we have modeled the interaction of the hairpin domain and the channel tunnel on a molecular level for AcrA and TolC as well as MexA and OprM, respectively. The model of two hairpin docking sites per TolC protomer corresponding with hexameric adaptor proteins was confirmed by disulfide cross-linking experiments. The role of this interaction for functional efflux pumps is discussed.     

Formation of cytoplasmic turns between two closely spaced transmembrane helices during membrane protein integration into the ER membrane

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a recurring structural element in integral membrane proteins, and may serve as a compact unit that inserts into the membrane en bloc. Previously, we have determined the propensities of the 20 natural amino acids, when present in the middle of a long hydrophobic stretch, to induce the formation of a helical hairpin with a lumenally exposed turn during membrane protein assembly into the endoplasmic reticulum membrane. Here, we present results from a similar set of measurements, but with the turn placed on the cytoplasmic side of the membrane. We find that a significantly higher number of turn-promoting residues need to be present to induce a cytoplasmic turn compared to a lumenal turn, and that, in contrast to the lumenal turn, the positively charged residues Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns.     

Lipid-destabilizing properties of the hydrophobic helices H8 and H9 from colicin E1

Colicins are toxic proteins produced by Escherichia coli that must cross the membrane to exert their activity. The lipid insertion of their pf domain is linked to a conformational change which enables the penetration of a hydrophobic hairpin. They provide useful models to more generally study insertion of proteins, channel formation and protein translocation in and across membranes. In this paper, we study the lipid-destabilizing properties of helices H8 and H9 forming the hydrophobic hairpin of colicin E1. Modelling analysis suggests that those fragments behave like tilted peptides. The latter are characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. Fluorescence techniques using labelled liposomes clearly show that H9, and H8 to a lesser extent, destabilize lipid particles, by inducing fusion and leakage. AFM assays clearly indicate that H8 and especially H9 induce membrane fragilization. Holes in the membrane are even observed in the presence of H9. This behaviour is close to what is seen with viral fusion peptides. Those results suggest that the peptides could be involved in the toroidal pore formation of colicin E1, notably by disturbing the lipids and facilitating the insertion of the other, more hydrophilic, helices that will form the pore. Since tilted, lipid-destabilizing fragments are also common to membrane proteins and to signal sequences, we suggest that tilted peptides should have an ubiquitous role in the mechanism of insertion of proteins into membranes.     

A semiempirical extension of polyelectrolyte theory to the treatment of oligoelectrolytes: Application to oligonucleotide helix-coil transitions

The interaction of counterions with a suitably long, charged oligomer appears susceptible to treatment in the context of polyelectrolyte theory by the introduction of an end-effect parameter that reflects the reduced association of counterions with the terminal regions of the oligo-ion. Use of a physically reasonable value for the end-effect parameter provides excellent agreement between theory and the experimental data of Elson, Scheffler, and Baldwin [J. Mol. Biol. 54 , 401–415 (1970)] on the dependences of melting temperature on salt concentration and chain length for a series of hairpin helices formed by d(TA) oligomers. The differences in behavior expected for hairpin, dimer, and oligomer-polymer helices are discussed. The salt dependence of the end-joining equilibrium investigated for λ DNA by Wang and Davidson [Cold Spring Harbor Symp. Quant. Biol. 33 , 409–415 (1968)] is treated as an oligomer–polymer interconversion. The dependence of equilibrium constant for this reaction on counterion concentration is in good agreement with that predicted by theory for an end-region totalling 24 nucleotides, the known length of the λ ends.     

BamA is required for autotransporter secretion

BackgroundIn Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the “passenger” domain) and a β-barrel that aids its export. While it is known that the folding and insertion of the β-barrel domain utilize the β-barrel assembly machinery (BAM) complex, how the passenger domain is secreted and folded across the membrane remains to be determined. The hairpin model states that passenger domain secretion occurs independently through the fully-formed and membrane-inserted β-barrel domain via a hairpin folding intermediate. In contrast, the BamA-assisted model states that the passenger domain is secreted through a hybrid of BamA, the essential subunit of the BAM complex, and the β-barrel domain of the autotransporter.MethodsTo ascertain the models' plausibility, we have used molecular dynamics to simulate passenger domain secretion for two autotransporters, EspP and YadA.ResultsWe observed that each protein's β-barrel is unable to accommodate the secreting passenger domain in a hairpin configuration without major structural distortions. Additionally, the force required for secretion through EspP's β-barrel is more than that through the BamA β-barrel.ConclusionsSecretion of autotransporters most likely occurs through an incompletely formed β-barrel domain of the autotransporter in conjunction with BamA.General significanceSecretion of virulence factors is a process used by practically all pathogenic Gram-negative bacteria. Understanding this process is a necessary step towards limiting their infectious capacity.     

DbpA is a region‐specific RNA helicase

DbpA is a DEAD‐box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA‐like catalytic core responsible for double‐helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site‐specific enzyme or region‐specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region‐specific or a site‐specific enzyme. Our data suggest that DbpA is a region‐specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C‐terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double‐helices in its vicinity. The only requirement for a double‐helix to serve as a DbpA substrate is for the double‐helix to be positioned within the catalytic core's grasp.     

Computational studies of colicin insertion into membranes: the closed state

Colicins are water-soluble toxins that, upon interaction with membranes, undergo a conformational change, insert, and form pores in them. Pore formation activity is localized in a bundle of 10 α-helices named the pore-forming domain (PFD). There is evidence that colicins attach to the membrane via a hydrophobic hairpin embedded in the core of the PFD. Two main models have been suggested for the membrane-bound state: penknife and umbrella, differing in regard to the orientation of the hydrophobic hairpin with respect to the membrane. The arrangement of the amphipathic helices has been described as either a compact three-dimensional structure or a two-dimensional array of loosely interacting helices on the membrane surface. Using molecular dynamics simulations with an implicit membrane model, we studied the structure and stability of the conformations proposed earlier for four colicins. We find that colicins are initially driven towards the membrane by electrostatic interactions between basic residues and the negatively charged membrane surface. They do not have a unique binding orientation, but in the predominant orientations the central hydrophobic hairpin is parallel to the membrane. In the inserted state, the estimated free energy tends to be lower for the compact arrangements of the amphipathic helix, but the more expanded ones are in better agreement with experimental distance distributions. The difference in energy between penknife and umbrella conformations is small enough for equilibrium to exist between them. Elongation of the hydrophobic hairpin helices and membrane thinning were found unable to produce stabilization of the transmembrane configuration of the hydrophobic hairpin.     

Structure of an atypical periplasmic adaptor from a multidrug efflux pump of the spirochete Borrelia burgdorferi

Periplasmic adaptor proteins are essential components of bacterial tripartite multidrug efflux pumps. Here we report the 2.35 Å resolution crystal structure of the BesA adaptor from the spirochete Borrelia burgdorferi solved using selenomethionine derivatized protein. BesA shows the archetypal linear, flexible, multi-domain architecture evident among proteobacteria and retains the lipoyl, β-barrel and membrane-proximal domains that interact with the periplasmic domains of the inner membrane transporter. However, it lacks the α-hairpin domain shown to establish extensive coiled-coil interactions with the periplasmic entrance helices of the outer membrane-anchored TolC exit duct. This has implications for the modelling of assembled tripartite efflux pumps.     

Transmembrane Helix Packing of ErbB/Neu Receptor in Membrane Environment: A Molecular Dynamics Study

Abstract

Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains.

Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor. Because experimental data do not distinguish clearly helix-helix packing, molecular dynamics (MD) simulations are used to investigate the energetic factors that drive Neu TM-TM interactions of the wild and the oncogenic receptor (Val664/Glu mutation) in DMPC or in POPC environments. MD results indicate that helix-lipid interactions in the bilayer core are extremely similar in the two environments and raise the role of the juxtamembrane residues in helix insertion and helix-helix packing. The TM domain shows a greater propensity to adopt a left-handed structure in DMPC, with helices in optimal position for strong inter-helical Hbonds induced by the Glu mutation. In POPC, the right-handed structure is preferentially formed with the participation of water in inter-helical Hbonds. The two structural arrangements of the NeuTM helices both with GG4 residue motif in close contact at the interface are permissible in the membrane environment. According to the hypothesis of a monomer-dimer equilibrium of the proteins it is likely that the bilayer imposes structural constraints that favor dimerization- competent structure responsible of the proper topology necessary for receptor activation.     

Coplanar and coaxial orientations of RNA bases and helices

Electrostatic interactions, base-pairing, and especially base-stacking dominate RNA three-dimensional structures. In an A-form RNA helix, base-stacking results in nearly perfect parallel orientations of all bases in the helix. Interestingly, when an RNA structure containing multiple helices is visualized at the atomic level, it is often possible to find an orientation such that only the edges of most bases are visible. This suggests that a general aspect of higher level RNA structure is a coplanar arrangement of base-normal vectors. We have analyzed all solved RNA crystal structures to determine the degree to which RNA base-normal vectors are globally coplanar. Using a statistical test based on the Watson-Girdle distribution, we determined that 330 out of 331 known RNA structures show statistically significant (p < 0.05; false discovery rate [FDR] = 0.05) coplanar normal vector orientations. Not surprisingly, 94% of the helices in RNA show bipolar arrangements of their base-normal vectors (p < 0.05). This allows us to compute a mean axis for each helix and compare their orientations within an RNA structure. This analysis revealed that 62% (208/331) of the RNA structures exhibit statistically significant coaxial packing of helices (p < 0.05, FDR = 0.08). Further analysis reveals that the bases in hairpin loops and junctions are also generally planar. This work demonstrates coplanar base orientation and coaxial helix packing as an emergent behavior of RNA structure and may be useful as a structural modeling constraint.     

Conformations of C-DNA in Agreement with Fiber X-ray and Infrared Dichroísm

Abstract

Different left handed double helices are proposed as models for DNA fibers in the C form. These conformations have geometrical parameters which agree well with fiber X-ray data and the orientations of their phosphate groups are in good agreement with infrared dichroísm results. A complete set of atomic coordinates and dihedral angles are given together with curves of calculated diffracted intensities. The present results are compared with experimental values and with the other C-DNA models.     

Insertion and organization within membranes of the delta-endotoxin pore-forming domain, helix 4-loop-helix 5, and inhibition of its activity by a mutant helix 4 peptide

The pore-forming domain of Bacillus thuringiensis Cry1Ac insecticidal protein comprises of a seven alpha-helix bundle (alpha1-alpha7). According to the "umbrella model," alpha4 and alpha5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, alpha4-loop-alpha5, its alpha4 and alpha5 helices, as well as mutant alpha4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the alpha4-loop-alpha5 hairpin is extremely active compared with the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes. Together with spectrofluorometric studies, we provide direct evidence for the role of alpha4-loop-alpha5 as the membrane-inserted pore-forming hairpin in which alpha4 and alpha5 line the lumen of the channel and alpha5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active alpha4 mutant peptide completely inhibits alpha4-loop-alpha5 pore formation, thus providing, to our knowledge, the first example that a mutated helix within a pore can function as an "immunity protein" by directly interacting with the segments that form the pore. This presents a potential means of interfering with the assembly and function of other membrane proteins as well.     

DNA Models for A,B, C and D Conformations Related to Fiber X-ray,Infrared and NMR Measurements

Abstract

A conformational analysis of the A, B, C and D DNA forms was made in order to establish molecular models presenting a good agreement with experimental data obtained from fiber X-ray, infrared linear dichroism and ³¹P NMR. The proposed models have been refined and do present good stereochemistry and optimized H-bond distances between bases associated with the Watson-Crick pairing. The DNA conformations proposed are a left handed double helix for the C form and right handed helices for A, B and D. Relations to conformational transitions between these forms are discussed.