Discovery of a significant,nontopological preference for antiparallel alignment of helices with parallel regions in sheets

To help elucidate the role of secondary structure packing preferences in protein folding, here we present an analysis of the packing geometry observed between alpha-helices and between alpha-helices and beta-sheets in 1316 diverse, nonredundant protein structures. Finite-length vectors were fit to the alpha-carbon atoms in each of the helices and strands, and the packing angle between the vectors, Omega, was determined at the closest point of approach within each helix-helix or helix-sheet pair. Helix-sheet interactions were found in 391 of the proteins, and the distributions of Omega values were calculated for all the helix-sheet and helix-helix interactions. The packing angle preferences for helix-helix interactions are similar to those previously observed. However, analysis of helix-strand packing preferences uncovered a remarkable tendency for helices to align antiparallel to parallel regions of beta-sheets, independent of the topological constraints or prevalence of beta-alpha-beta motifs in the proteins. This packing angle preference is significantly diminished in helix interactions involving mixed and antiparallel beta-sheets, suggesting a role for helix-sheet dipole alignment in guiding supersecondary structure formation in protein folding. This knowledge of preferred packing angles can be used to guide the engineering of stable protein modules.     

Unique stabilizing interactions identified in the two-stranded alpha-helical coiled-coil: crystal structure of a cortexillin I/GCN4 hybrid coiled-coil peptide

We determined the 1.17 A resolution X-ray crystal structure of a hybrid peptide based on sequences from coiled-coil regions of the proteins GCN4 and cortexillin I. The peptide forms a parallel homodimeric coiled-coil, with C(alpha) backbone geometry similar to GCN4 (rmsd value 0.71 A). Three stabilizing interactions have been identified: a unique hydrogen bonding-electrostatic network not previously observed in coiled-coils, and two other hydrophobic interactions involving leucine residues at positions e and g from both g-a' and d-e' interchain interactions with the hydrophobic core. This is also the first report of the quantitative significance of these interactions. The GCN4/cortexillin hybrid surprisingly has two interchain Glu-Lys' ion pairs that form a hydrogen bonding network with the Asn residues in the core. This network, which was not observed for the reversed Lys-Glu' pair in GCN4, increases the combined stability contribution of each Glu-Lys' salt bridge across the central Asn15-Asn15' core to approximately 0.7 kcal/mole, compared to approximately 0.4 kcal mole(-1) from a Glu-Lys' salt bridge on its own. In addition to electrostatic and hydrogen bonding stabilization of the coiled-coil, individual leucine residues at positions e and g in the hybrid peptide also contribute to stability by 0.7 kcal/mole relative to alanine. These interactions are of critical importance to understanding the stability requirements for coiled-coil folding and in modulating the stability of de novo designed macromolecules containing this motif.     

Core side-chain packing and backbone conformation in Lpp-56 coiled-coil mutants

Native proteins exhibit precise geometric packing of atoms in their hydrophobic interiors. Nonetheless, controversy remains about the role of core side-chain packing in specifying and stabilizing the folded structures of proteins. Here we investigate the role of core packing in determining the conformation and stability of the Lpp-56 trimerization domain. The X-ray crystal structures of Lpp-56 mutants with alanine substitutions at two and four interior core positions reveal trimeric coiled coils in which the twist of individual helices and the helix-helix spacing vary significantly to achieve the most favored superhelical packing arrangement. Introduction of each alanine "layer" into the hydrophobic core destabilizes the superhelix by 1.4 kcal mol(-1). Although the methyl groups of the alanine residues pack at their optimum van der Waals contacts in the coiled-coil trimer, they provide a smaller component of hydrophobic interactions than bulky hydrophobic side-chains to the thermodynamic stability. Thus, specific side-chain packing in the hydrophobic core of coiled coils are important determinants of protein main-chain conformation and stability.     

Protein destabilization by electrostatic repulsions in the two-stranded alpha-helical coiled-coil/leucine zipper.

The destabilizing effect of electrostatic repulsions on protein stability has been studied by using synthetic two-stranded alpha-helical coiled-coils as a model system. The native coiled-coil consists of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain disulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled-coil structure at 20 degrees C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 value of 6.1 M. Four mutant coiled-coils were designed to contain one or two Glu substitutions for Gln per polypeptide chain. The resulting coiled-coils contained potential i to i' + 5 Glu-Glu interchain repulsions (denoted as peptide E2(15,20)), i to i' + 2 Glu-Glu interchain repulsions (denoted E2(20,22)), or no interchain ionic interactions (denoted E2(13,22) and E1(20)). The stabilities of the coiled-coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 degrees C in the presence and absence of an interchain disulfide bridge. At pH 7, in the presence of urea, the stabilities of E2(13,22) and E2(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu-Glu repulsion in the E2(20,22) coiled-coil does not occur. In contrast, the mutant E2(15,20) is substantially less stable than E2(13,22) or E2(15,20) by 0.9 kcal/mol due to the presence of two i to i' + 5 interchain Glu-Glu repulsions, which destabilize the coiled-coil by 0.45 kcal/mol each. At pH 3 the coiled-coils were found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed-phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chains present at low pH are significantly more hydrophobic than Gln side chains which are in turn more hydrophobic than the ionized Glu side chains present at neutral pH. The protonated Glu residues increase the hydrophobicity of the coiled-coil interface leading to higher coiled-coil stability. The guanidine hydrochloride results at pH 7 show similar stabilities between the native and mutant coiled-coils indicating that guanidine hydrochloride masks electrostatic repulsions due to its ionic nature and that Glu and Gln in the e and g positions of the heptad repeat have very similar effects on coiled-coil stability in the presence of GdnHCl.     

Self-assembly of coiled-coil tetramers in the 1.40 A structure of a leucine-zipper mutant

The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel double-stranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.     

Structure of a designed, right-handed coiled-coil tetramer containing all biological amino acids

The previous design of an unprecedented family of two-, three-, and four-helical, right-handed coiled coils utilized nonbiological amino acids to efficiently pack spaces in the oligomer cores. Here we show that a stable, right-handed parallel tetrameric coiled coil, called RH4B, can be designed entirely using biological amino acids. The X-ray crystal structure of RH4B was determined to 1.1 Angstrom resolution using a designed metal binding site to coordinate a single Yb(2+) ion per 33-amino acid polypeptide chain. The resulting experimental phases were particularly accurate, and the experimental electron density map provided an especially clear, unbiased view of the molecule. The RH4B structure closely matched the design, with equivalent core rotamers and an overall root-mean-square deviation for the N-terminal repeat of the tetramer of 0.24 Angstrom. The clarity and resolution of the electron density map, however, revealed alternate rotamers and structural differences between the three sequence repeats in the molecule. These results suggest that the RH4B structure populates an unanticipated variety of structures.     

Automated design of the surface positions of protein helices.

Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of alpha helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4-p1 were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures from 69-72 degrees C, over 12 degrees C higher than GCN4-p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 degrees C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.     

Optimal bundling of transmembrane helices using sparse distance constraints

We present a two-step approach to modeling the transmembrane spanning helical bundles of integral membrane proteins using only sparse distance constraints, such as those derived from chemical cross-linking, dipolar EPR and FRET experiments. In Step 1, using an algorithm, we developed, the conformational space of membrane protein folds matching a set of distance constraints is explored to provide initial structures for local conformational searches. In Step 2, these structures refined against a custom penalty function that incorporates both measures derived from statistical analysis of solved membrane protein structures and distance constraints obtained from experiments. We begin by describing the statistical analysis of the solved membrane protein structures from which the theoretical portion of the penalty function was derived. We then describe the penalty function, and, using a set of six test cases, demonstrate that it is capable of distinguishing helical bundles that are close to the native bundle from those that are far from the native bundle. Finally, using a set of only 27 distance constraints extracted from the literature, we show that our method successfully recovers the structure of dark-adapted rhodopsin to within 3.2 A of the crystal structure.     

Sequence and conformational preferences at termini of α‐helices in membrane proteins: Role of the helix environment

α‐helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α‐helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C‐termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α‐helices in a high‐resolution dataset of integral α‐helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C‐termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near‐helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420–3436. © 2014 Wiley Periodicals, Inc.     

Crystal structure of the coiled-coil domain of Drosophila TRIM protein Brat

Drosophila brain tumor (Brat) is a translational repressor belonging to the tripartite motif (TRIM) protein superfamily. During the asymmetric division of Drosophila neuroblasts, Brat localizes at the basal cortex via direct interaction with the scaffolding protein Miranda (Mira), and segregates into the basal ganglion mother cells after cell division. It was previously reported that both the coiled-coil (CC) and NHL domains of Brat are required for the interaction with Mira, but the underlying structural basis is elusive. Here, we determine the crystal structure of Brat-CC domain (aa 376-511) at 2.5 Å, showing that Brat-CC forms an elongated antiparallel dimer through an unconventional CC structure. The dimeric assembly in Brat-CC structure is similar to its counterparts in other TRIM proteins, but Brat-CC also exhibits some distinct structural features. We also demonstrate that the CC domain could not bind Mira by its own, neither does the isolated NHL domain of Brat. Rather, Brat binds to Mira through the CC-NHL domain tandem, indicating that the function of the CC domain is to assemble Brat-NHL in dimeric form, which is necessary for Mira binding.     

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.     

A new perspective on analysis of helix-helix packing preferences in globular proteins

For many years, statistical analysis of protein databanks has led to the belief that the steric compatibility of helix interfaces may be the source of observed preferences for particular angles between neighboring helices. Several elegant models describing how side chains on helices can interdigitate without steric clashes were able to account quite reasonably for the observed distributions. However, it was later recognized that the 'bare' measured angle distribution should be corrected to avoid statistical bias.12 Disappointingly, the rescaled distributions dramatically lost their similarity with theoretical predictions, casting doubts on the validity of the geometrical assumptions and models. In this article, we elucidate a few points concerning the proper choice of a random reference distribution. In particular we demonstrate the need for corrections induced by unavoidable uncertainties in determining whether two helices are in face-to-face contact or not and their relative orientations. By using this new rescaling, we show that 'true' packing angle preferences are well described by regular packing models, thus proving that preferential angles between contacting helices do exist.     

Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145-amino acid residues with extensive alpha-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.     

TMSEG: Novel prediction of transmembrane helices

Transmembrane proteins (TMPs) are important drug targets because they are essential for signaling, regulation, and transport. Despite important breakthroughs, experimental structure determination remains challenging for TMPs. Various methods have bridged the gap by predicting transmembrane helices (TMHs), but room for improvement remains. Here, we present TMSEG, a novel method identifying TMPs and accurately predicting their TMHs and their topology. The method combines machine learning with empirical filters. Testing it on a non‐redundant dataset of 41 TMPs and 285 soluble proteins, and applying strict performance measures, TMSEG outperformed the state‐of‐the‐art in our hands. TMSEG correctly distinguished helical TMPs from other proteins with a sensitivity of 98 ± 2% and a false positive rate as low as 3 ± 1%. Individual TMHs were predicted with a precision of 87 ± 3% and recall of 84 ± 3%. Furthermore, in 63 ± 6% of helical TMPs the placement of all TMHs and their inside/outside topology was correctly predicted. There are two main features that distinguish TMSEG from other methods. First, the errors in finding all helical TMPs in an organism are significantly reduced. For example, in human this leads to 200 and 1600 fewer misclassifications compared to the second and third best method available, and 4400 fewer mistakes than by a simple hydrophobicity‐based method. Second, TMSEG provides an add‐on improvement for any existing method to benefit from. Proteins 2016; 84:1706–1716. © 2016 Wiley Periodicals, Inc.     

Non-randomness in side-chain packing: the distribution of interplanar angles

We analyze the distributions of interplanar angles between interacting side chains with well-defined planar regions, to see whether these distributions correspond to random packing or alternatively show orientational preferences. We use a non-homologous set of 79 high-resolution protein chain structures to show that the observed distributions are significantly different from the sinusoidal one expected for random packing. Overall, we see a relative excess of small angles and a paucity of large interplanar angles; the difference between the expected and observed distributions can be described as a shift of 5% of the interplanar angles from large (≥60°) to small (<30°) values. By grouping the residue pairs into categories based on chemical similarity, we find that some categories have very non-sinusoidal interplanar angle distributions, whereas other categories have distributions that are close to sinusoidal. For a few categories, observed deviations from a sinusoidal distribution can be explained by the electrostatic anisotropy of the isolated pair potential energy. In other cases, the observed distributions reflect the longer range effects of different possible interaction geometries. In particular, geometries that disrupt external hydrogen bonding are disfavored. Proteins 29:370–380, 1997. © 1997 Wiley-Liss, Inc.     

Extended knobs-into-holes packing in classical and complex coiled-coil assemblies

This year marks the 50th anniversary of Crick’s seminal paper on the packing of α-helices into coiled-coil structures. The central tenet of Crick’s work is the interdigitation of side chains, which directs the helix–helix interactions; so called knobs-into-holes packing. Subsequent determinations of coiled-coil-protein sequences and structures confirmed the key features of Crick’s model and established it as a fundamental concept in structural biology. Recently, we developed a program, SOCKET, to recognise knobs-into-holes packing in protein structures, which we applied to the Protein Data Bank to compile a database of coiled-coil structures. In addition to classic structures, the database reveals 4-helix bundles and larger helical assemblies. Here, we describe how the more-complex structures can be understood by extending Crick’s principles for classic coiled coils. In the simplest case, each helix of a 2-stranded structure contributes a single seam of (core) knobs-into-holes to the helical interface. 3-, 4-, and 5-Stranded structures, however, are best considered as rings of helices with cycles of knobs-into-holes. These higher-order oligomers make additional (peripheral) knobs-into-holes that broaden the helical contacts. Combinations of core and peripheral knobs may be assigned to different sequence repeats offset within the same helix. Such multiple repeats lead to multi-faceted helices, which explain structures above dimers. For instance, coiled-coil oligomer state correlates with the offset of the different repeats along a sequence. In addition, certain multi-helix assemblies can be considered as conjoined coiled coils in which multi-faceted helices participate in more than one coiled-coil motif.     

Design,selection, and characterization of a split chorismate mutase

Split proteins are versatile tools for detecting protein–protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical‐bundle CM from Methanococcus jannaschii into a short N‐terminal helix and a 3‐helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild‐type like CM activity. Owing to the availability of a well characterized selection system, the simple 4‐helix bundle topology, and the small size of the N‐terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein–protein interactions.     

Characterization of a new four-chain coiled-coil: influence of chain length on stability.

Limited information is available on inherent stabilities of four-chain-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain alpha-helical chains 21, 28, and 35 amino acids in length are 3.1 x 10(-12), 6.7 x 10(-23), and 1.0 x 10(-38) M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 microM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled-coil formation in light of the work presented here.     

Design and characterization of the anion-sensitive coiled-coil peptide.

As a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled-coil. The interface between the coils consisted of hydrophobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide-linked dimer in which the two peptides were linked in parallel by the two disulfide bonds located at the N and C terminals, a cyclic monomer with an intramolecular disulfide bond was also obtained. On the basis of CD and 1H-NMR, the conformational stabilities of these isomers and several reference peptides were examined. Whereas all these peptides were unfolded in the absence of salt at pH 4.7 and 20 degrees C, the addition of NaClO4 cooperatively stabilized the alpha-helical conformation. The crosslinking of the peptides by disulfide bonds significantly decreased the midpoint salt concentration of the transition. The 1H-NMR spectra in the presence of NaClO4 suggested that, whereas the disulfide-bonded dimer assumed a native-like conformation, the cyclic monomer assumed a molten globule-like conformation with disordered side chains. However, the cyclic monomer exhibited cooperative transitions against temperature and Gdn-HCl that were only slightly less cooperative than those of the disulfide-bonded parallel dimer. These results indicate that the charge repulsion critically destabilizes the native-like state as well as the molten globule-like state, and that the solvent-dependent charge repulsion may be useful for controlling the conformation of designed peptides.