Molecular Dynamics Simulations of β-turn Forming Tetra- and Hexapeptides

Abstract

It was previously shown that the structural ensemble of model peptides DDKG and GKDG (H. Ishii et al. Biopolymers 24, 2045–2056, 1985), DEKS (A. Otter et al. J. Biomol. Struct. Dyn. 7, 455–476, 1989) NPGQ (F. R. Carbone et al. Int. J. Pept. Protein. Res. 26, 498–508, 1985), SALN (H. Santa et al. J. Biomol. Struct. Dyn. 16, 1033–1041, 1999), SYPFDV and SYPYDV (J. Yao et al. J. Mol. Biol. 243, 736–753, 1994), VP^DAH and VP^DSH (B. Imperiali et al. J. Am. Chem. Soc. 114, 3182–3188, 1992) in solution contains a significant—or in some cases dominant—proportion of β-turn conformation. In this study, a protein database was searched for the above, unprotected sequences which incorporate only L-amino acid residues. Simulated annealing and 25 ns MD simulations of structures were also performed. The DSSP and STRIDE secondary structure-assigning algorithms and clustering were used to analyze trajectories and i, i+3 hydrogen bonds were also sought. The DSSP analysis showed a fluctuation between β-turn and random meander structure, although bend structures were not detected because of the insufficient length of peptide chains. This alternating trend was confirmed when the STRIDE algorithm was used to analyze trajectories, but STRIDE assigned more turn structures. The population of the strongest clusters was above 40% and the middle structures adopted β-turn structure for most sequences. These results are in good agreement with previous experimental results and support the idea of the ultra-marginal stability of turns in the absence of stabilizing long-range interactions of the neighboring segments of a polypeptide chain. However, interactions between the side-chains in tetrapeptides could also contribute to turn stability and result in unusual stability in some cases. Our observations suggest that such interactions are the consequence rather than the driving force of turn formation.     

Computational assessment of folding energy landscapes in heterodimeric coiled coils

The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs‐into‐holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.     

Exploring alternate states and oligomerization preferences of coiled‐coils by de novo structure modeling

Homomeric coiled‐coils can self‐assemble into a wide range of structural states with different helix topologies and oligomeric states. In this study, we have combined de novo structure modeling with stability calculations to simultaneously predict structure and oligomeric states of homomeric coiled‐coils. For dimers an asymmetric modeling protocol was developed. Modeling without symmetry constraints showed that backbone asymmetry is important for the formation of parallel dimeric coiled‐coils. Collectively, our results demonstrate that high‐resolution structure of coiled‐coils, as well as parallel and antiparallel orientations of dimers and tetramers, can be accurately predicted from sequence. De novo modeling was also used to generate models of competing oligomeric states, which were used to compare stabilities and thus predict the native stoichiometry from sequence. In a benchmark set of 33 coiled‐coil sequences, forming dimers to pentamers, up to 70% of the oligomeric states could be correctly predicted. The calculations demonstrated that the free energy of helix folding could be an important factor for determining stability and oligomeric state of homomeric coiled‐coils. The computational methods developed here should be broadly applicable to studies of sequence‐structure relationships in coiled‐coils and the design of higher order assemblies with improved oligomerization specificity. Proteins 2015; 83:235–247. © 2014 Wiley Periodicals, Inc.     

Thermodynamic model for the stabilization of trigonal thiolato mercury(II) in designed three-stranded coiled coils.

A thermodynamic model is presented that describes the binding of Hg(II) to de novo designed peptides, Tri L9C and Baby L9C, which were derived from the Tri family. The Tri peptides are based on the parent sequence Ac-NH-G(LKALEEK)(x)()G-CONH(2) and are known to form two-stranded coiled coils at low pH (pH <4) and three-stranded coiled coils at high pH (pH >7). Tri L9C (x = 4) contains a four heptad repeat sequence with cysteine in position 9 and leucines in the other a and d positions; Baby L9C (x = 3), which also has a cysteine in position 9 but is one heptad shorter than Tri L9C, was designed to form less stable helical coiled coils in solution. The free energies of coiled coil formation for Tri, Tri L9C, Baby Tri, and Baby L9C at pH 2.5 and 8.5 were determined by guanidinium denaturation titrations; Tri L9C was observed to be highly helical in the absence of denaturant at pH 8.5 while Baby L9C contained <20% helical content at pH 8.5, indicating a weakly associated or unassociated coiled coil. Size-exclusion chromatography (SEC) verified that Baby L9C was a monomer at pH 8.5. The helicity of Baby L9C was induced by addition of HgCl(2). The subsequent formation of a trigonal thiolato Hg(II) in the interior of a three-stranded coiled coil was verified by the presence of a characteristic HgS(3) UV band at 248 nm. Titrations of Tri L9C and Baby L9C into solutions of HgCl(2) at pH values between 7 and 9 were performed to extract binding constants. Global fits to the data employed a mechanism that involved initial binding of mercury to the peptides forming a two-stranded coiled coil with linear thiolato Hg(II) at [peptide]/[Hg] <2, followed by addition of a more weakly associated third helix to generate a three-stranded coiled coil. This mechanism would require the deprotonation of the third cysteine thiol to generate the trigonal thiolato Hg(II) at pH >7.5 [the pK(a) of the cysteine thiol in the presence of Hg(II)]. Support for this mechanism was given by the observation of a three-stranded coiled coil by SEC in a solution of Tri L9C at pH 7.0.     

Structure and stability of the C‐terminal helical bundle of the E. coli mechanosensitive channel of large conductance

The crystal structure of the cytoplasmic domain (CTD) from the mechanosensitive channel of large conductance (MscL) in E. coli has been determined at 1.45 Å resolution. This domain forms a pentameric coiled coil similar to that observed in the structure of MscL from M. tuberculosis and also found in the cartilage oligomeric matrix protein (COMPcc). It contains canonical hydrophobic and atypical ionic interactions compared to previously characterized coiled coil structures. Thermodynamic analysis indicates that while the free EcMscL‐CTD is less stable than other coiled coils, it is likely to remain folded in context of the full‐length channel.     

Effects of five-membered ring amino acid incorporation into peptides for coiled coil formation

A five-membered ring amino acid (Ac₅c), the peptides of which exhibit a preference for helical secondary structures, was introduced into peptides for the purpose of designing coiled coil peptides with high binding affinities. We prepared five types of peptides containing Ac₅c with different numbers or at different positions. The incorporation of Ac₅c into peptides enhanced their α-helicities; however, in contrast to our expectations, it did not result in stable coiled coil formation. The structures of side chains in hydrophobic amino acids, not α-helicities appeared to be important for stable hydrophobic interactions between peptides. Although we were unable to develop coiled coil peptides with high binding affinities, the present results will be useful for designing novel coiled coil peptides.     

Spectroscopy-Based Modelling of the 3D Structure of the β Subunit of the High Affinity IgE Receptor

Abstract

The high affinity IgE receptor, possesses a tetrameric structure. The 243 residue β subunit is a polytopic protein with four hydrophobic membrane-spanning segments, whereas the individual α and γ subunits are bitopic proteins each containing one transmembrane domain in their monomeric form. In the proposed topographical model (Blank et al., 1989), the four trans-membrane α helices of the β subunit are connected by three loop sequences.

To study the individual subunits and intact receptor, this membrane protein was divided into domains such as its loop peptides, cytoplasmic peptides and transmembrane helices according to Blank et al., 1989. The 3D structure of the synthesized loop peptides and cytoplasmic peptides were calculated; CD and/or NMR data were used as appropriate to generate the resultant structures which were then used as data basis for the higher level calculations.

The four individual transmembrane helices of the β subunit were characterised, first of all, by mapping the relative lipophilicity of their surfaces using lipophilic probes. A second procedure, docking of the individual helices in pairs, was used to predict helix–helix interactions.

The data on the relative lipophilicity of the surfaces as well as the surfaces that favoured helix–helix interactions were used in combination with the spectroscopy-based structures of the loops and cytoplasmic domains to calculate via molecular dynamics, the helix arrangement and 3D structure of the β subunit of the high affinity IgE receptor. In the final analysis, the molecular simulations yielded two structures of the β subunit, which should form a basis for the modelling of the whole high affinity IgE receptor.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies.     

Structural mapping of the coiled‐coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins

The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N‐ and C‐ terminal regions pack against one another to form a globular ATPase domain. This “head” domain is connected to a central, globular, “hinge” or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50‐nm coiled‐coil domain of MukB, the divergent SMC protein found in γ‐proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled‐coil domain. We find that, in contrast to the relatively complicated coiled‐coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled‐coil interruptions. Near the middle of the domain is a break in coiled‐coil structure in which there are three more residues on the C‐terminal strand than on the N‐terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled‐coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled‐coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. Proteins 2015; 83:1027–1045. © 2015 Wiley Periodicals, Inc.     

Electrostatic contribution to the thermodynamic and kinetic stability of the homotrimeric coiled coil Lpp-56: A computational study

The protein moiety of the Braun's E. coli outer membrane lipoprotein (Lpp-56) is an attractive object of biophysical investigation in several aspects. It is a homotrimeric, parallel coiled coil, a class of coiled coils whose stability and folding have been studied only occasionally. Lpp-56 possesses unique structural properties and exhibits extremely low rates of folding and unfolding. It is natural to ask how the specificity of the structure determines the extraordinary physical chemical properties of this protein. Recently, a seemingly controversial data on the stability and unfolding rate of Lpp-56 have been published (Dragan et al., Biochemistry 2004;43: 14891-14900; Bjelic et al., Biochemistry 2006;45:8931-8939). The unfolding rate constant measured using GdmCl as the denaturing agent, though extremely low, was substantially higher than that obtained on the basis of thermal unfolding. If this large difference arises from the effect of screening of electrostatic interactions induced by GdmCl, electrostatic interactions would appear to be an important factor determining the unusual properties of Lpp-56. We present here a computational analysis of the electrostatic properties of Lpp-56 combining molecular dynamics simulations and continuum pK calculations. The pH-dependence of the unfolding free energy is predicted in good agreement with the experimental data: the change in DeltaG between pH 3 and pH 7 is approximately 60 kJ mol(-1). The results suggest that the difference in the stability of the protein observed using different experimental methods is mainly because of the effect of the reduction of electrostatic interactions when the salt (GdmCl) concentration increases. We also find that the occupancy of the interhelical salt bridges is unusually high. We hypothesize that electrostatic interactions, and the interhelical salt bridges in particular, are an important factor determining the low unfolding rate of Lpp-56.     

Helicity of short E-R/K peptides

Understanding the secondary structure of peptides is important in protein folding, enzyme function, and peptide‐based drug design. Previous studies of synthetic Ala‐based peptides (>12 a.a.) have demonstrated the role for charged side chain interactions involving Glu/Lys or Glu/Arg spaced three (i, i + 3) or four (i, i + 4) residues apart. The secondary structure of short peptides (<9 a.a.), however, has not been investigated. In this study, the effect of repetitive Glu/Lys or Glu/Arg side chain interactions, giving rise to E‐R/K helices, on the helicity of short peptides was examined using circular dichroism. Short E‐R/K–based peptides show significant helix content. Peptides containing one or more E‐R interactions display greater helicity than those with similar E‐K interactions. Significant helicity is achieved in Arg‐based E‐R/K peptides eight, six, and five amino acids long. In these short peptides, each additional i + 3 and i + 4 salt bridge has substantial contribution to fractional helix content. The E‐R/K peptides exhibit a strongly linear melt curve indicative of noncooperative folding. The significant helicity of these short peptides with predictable dependence on number, position, and type of side chain interactions makes them an important consideration in peptide design.     

Effect of side chain length on intrahelical interactions between carboxylate- and guanidinium-containing amino acids

The charge-containing hydrophilic functionalities of encoded charged amino acids are linked to the backbone via different numbers of hydrophobic methylenes, despite the apparent electrostatic nature of protein ion pairing interactions. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on ion pairing interactions, α-helical peptides containing Zbb–Xaa (i, i + 3), (i, i + 4) and (i, i + 5) (Zbb = carboxylate-containing residues Aad, Glu, Asp in decreasing length; Xaa = guanidinium residues Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by circular dichroism spectroscopy (CD). The helicity of Aad- and Glu-containing peptides was similar and mostly pH independent, whereas the helicity of Asp-containing peptides was mostly pH dependent. Furthermore, the Arg-containing peptides consistently exhibited higher helicity compared to the corresponding Agp-, Agb-, and Agh-containing peptides. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb–Xaa (i, i + 3) and (i, i + 4) interactions mainly involved the χ ₁ dihedral combinations (g+, g+) and (g?, g+), respectively. These low energy conformations were also observed in intrahelical Asp–Arg and Glu–Arg salt bridges of natural proteins. Accordingly, Asp and Glu provides variation in helix characteristics associated with Arg, but Aad does not provide features beyond those already delivered by Glu. Importantly, nature may have chosen the side chain length of Arg to support helical conformations through inherent high helix propensity coupled with stabilizing intrahelical ion pairing interactions with the carboxylate-containing residues.     

Skeletal keratan sulphate structural analysis using keratanase II digestion followed by high-performance anion-exchange chromatography

A semi-quantitative fingerprinting method has been developedfor the structural analysis of skeletal keratan sulphates. Thisinvolves the digestion of the parent keratan sulphate chainswith the enzyme keratanase II (Bacillus sp.), followed by reductionof the resulting oligosaccharides with sodium borohydride andchromatography on a Dionex AS4A-SC column. This column has beencalibrated for the elution positions of 26 previously characterizedoligosaccharides (Brown et al., Biochemistry, 33, 4836–4846,1994; Brown et al., Eur. J. Biochem., 224, 281–308, 1994).The technique permits sample analysis with pulsed electrochemicaldetection (sensitive to     

Local destabilization of the tropomyosin coiled coil gives the molecular flexibility required for actin binding

Tropomyosin, a coiled coil protein that binds along the length of actin filaments, contains 40 uninterrupted heptapeptide repeats characteristic of coiled coils. Yet, it is flexible. Regions of tropomyosin that may be important for binding to the filament and for interacting with troponin deviate from canonical coiled coil structure in subtle ways, altering the local conformation or energetics without interrupting the coiled coil. In a region rich in interface alanines (an Ala cluster), the chains pack closer than in canonical coiled coils, and are staggered, resulting in a bend [Brown et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8496-8501]. Brown et al. suggested that bends at alanine clusters allow tropomyosin to wind on the actin filament helix. Another explanation is that local destabilization of the coiled coil, rather than close packing of the chains at Ala clusters per se, allows flexibility. Changing three Ala residues to canonical interface residues, A74L-A78V-A81L, greatly stabilized tropomyosin, measured using circular dichroism and differential scanning calorimetry, and reduced actin affinity >10-fold. Normal actin affinity and stability were restored in a mutant A74Q-A78N-A81Q that mimicked the stability of the Ala cluster but not the close packing of the chains. Analysis and modeling of comparable mutations introduced closer to the N-terminus show that the effects on stability and function depend on context. Models based on tropomyosin crystal structures give insight into possible effects of the mutations on the structure. We conclude that the significance of the Ala clusters in allowing flexibility of tropomyosin is stability-driven.     

Sequence Conservation in Plasmodium falciparum α-Helical Coiled Coil Domains Proposed for Vaccine Development

Background

The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to α-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative α-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB.

Methodology/Principal Findings

14 α-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 α-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative α-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for α-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved α-helical coiled coil motifs.

Conclusion/Significance

We conclude that the selection of α-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation.     

Interaction of transmembrane helices by a knobs-into-holes packing characteristic of soluble coiled coils

Membrane-embedded protein domains frequently exist as α-helical bundles, as exemplified by photosynthetic reaction centers, bacteriorhodopsin, and cytochrome C oxidase. The sidechain packing between their transmembrane helices was investigated by a nearest-neighbor analysis which identified sets of interfacial residues for each analyzed helix–helix interface. For the left-handed helix–helix pairs, the interfacial residues almost exclusively occupy positions a, d, e, or g within a heptad motif (abcdefg) which is repeated two to three times for each interacting helical surface. The connectivity between the interfacial residues of adjacent helices conforms to the knobs-into-holes type of sidechain packing known from soluble coiled coils. These results demonstrate on a quantitative basis that the geometry of sidechain packing is similar for left-handed helix–helix pairs embedded in membranes and coiled coils of soluble proteins. The transmembrane helix–helix interfaces studied are somewhat less compact and regular as compared to soluble coiled coils and tolerate all hydrophobic amino acid types to similar degrees. The results are discussed with respect to previous experimental findings which demonstrate that specific interactions between transmembrane helices are important for membrane protein folding and/or oligomerization. Proteins 31:150–159, 1998. © 1998 Wiley-Liss, Inc.     

Cysteine 265 Is in the Active Site of, But Is Not Essential for Catalysis by tRNA-Guanine Transglycosylase (TGT) from Escherichia coli

Site-directed mutagenesis and X-ray absorption spectroscopy studies have previously shown that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is a zinc metalloprotein and identified the enzymic ligands to the zinc [Chong et al. (1995), Biochemistry 34, 3694–3701; Garcia et al. (1966), Biochemistry 35, 3133–3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. The present report demonstrates that TGT is inactivated by treatment with thiol reagents (e.g., DTNB, MMTS, and N-ethylmaleimide). Further, this inactivation is shown to be due to modification of cysteine 265. The kinetic parameters for the mutants TGT (C265A) and TGT (C265S), however, suggest that this residue is not performing a critical role in the TGT reaction. We conclude that cysteine 265 is in the active site of TGT, but is not performing a critical catalytic function. This conclusion is supported by the recent determination of the X-ray crystal structure of the TGT from Zymomonas mobilis [Romier et al. (1966), EMBO J. 15, 2850–2857], which reveals that the residue corresponding to cysteine 265 is distant from the putative catalytic site, but is in the middle of a region of the enzyme surface proposed to bind tRNA.