Molecular modeling indicates that homodimers form the basis for intermediate filament assembly from human and mouse epidermal keratins

Mammalian epidermal keratin molecules adopt rod-shaped conformations that aggregate to form cytoplasmic intermediate filaments. To investigate these keratin conformations and the basis for their patterns of molecular association, graphical methods were developed to relate known amino acid sequences to probable spacial configurations. The results support the predominantly α-helical conformation of keratin chains, interrupted by short non-α-helical linkages. However, it was found that many of the linkages have amino acid sequences typical of β-strand conformations. Space-filling atomic models revealed that the β-strand sequences would permit the formation of 2-chain and 4-chain cylindrical β-helices, fully shielding the hydrophobic amino acid chains that alternate with hydrophilic residues in these sequences. Because of the locations of the β-helical regions in human and mouse stratum corneum keratin chains, only homodimers of the keratins could interact efficiently to form 2-chain and 4-chain β-helices. Tetramers having the directions and degrees of overlap of constituent dimers that have been identified by previous investigators are also predicted from the interactions of β-helical motifs. Heterotetramers formed from dissimilar homodimers could combine, through additional β-helical structures, to form higher oligomers having the dimensions seen in electron microscopic studies. Previous results from chemical crosslinking studies can be interpreted to support the concept of homodimers rather than heterodimers as the basis for keratin filament assembly. © 1995 Wiley-Liss, Inc.     

Chemical cross-linking between lysine groups in vimentin oligomers is dependent on local peptide conformations

Previous studies have shown that cytoplasmic intermediate filaments, other than the keratins, are each constructed from a single type of polypeptide chain. Studies involving chemical crosslinking between lysine groups have shown that assembly of the filaments begins with the formation of dimers in which the peptide chains are parallel and in exact register, and that these dimers further associate in antiparallel patterns having specific degrees of overlap. In the present study, molecular modeling of the conformations of vimentin molecules indicated that lysine side chains in identical positions in regions of α-helix in parallel chains might be unable to be linked because they are on opposite sides of the coiled coil hydrophobic core. Examination of published data on chemical crosslinking of lysines in vimentin confirmed that there were no instances of linkage within dimers between the nine pairs of identical lysines that lie more than one position within α-helical regions in parallel chains. Even among linkages that apparently were between dimers, only one of the 11 linkage products identified involved lysines that were both within an α-helical region. In 10 of the 11 identified linkages between dimers, one or both of the linked lysines were in regions of random coil conformation. These results of molecular modeling indicate that relative motion between polypeptide chains in oligomers of intermediate filament proteins is not sufficient to overcome an orientation of lysine groups that is unfavorable for their chemical linkage. This finding supports the interpretations of keratin cross-linking data indicating that parallel homodimers are the basis for keratin intermediate filament assembly. © 1996 Wiley-Liss, Inc.     

Molten globule state of equine β-Lactoglobulin

The acid-unfolded state of equine β-lactoglobulin was characterized by means of circular dichroism, nuclear magnetic resonance, analytical gel-filtration chromatography, and analytical centrifugation. The acid-unfolded state of equine β-lactoglobulin has a substantial secondary structure as shown by the far-ultraviolet circular dichroism spectrum but lacks persistent tertiary packing of the side chains as indicated by the near-ultraviolet circular dichroism and nuclear magnetic resonance spectra. It is nearly as compact as the native conformation as shown by the gel filtration and sedimentation experiments, and it has the exposed hydrophobic surface as indicated by its tendency to aggregate. All of these characteristics indicate that the acid-unfolded state of equine β-lactoglobulin is a molten globule state. The α helix content in the acid-unfolded state, which has been estimated from the circular dichroism spectrum, is larger than that in the native state, suggesting the presence of nonnative α helices in the molten globule state. This result suggests the generality of the intermediate with nonnative α helices during the folding of proteins having the β-clam fold. © 1997 Wiley-Liss Inc.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Molecular simulations of β‐lactoglobulin complexed with fatty acids reveal the structural basis of ligand affinity to internal and possible external binding sites

The interaction of saturated fatty acids of different length (C8:0 to C18:0) with β‐lactoglobulin (βLG) was investigated by molecular dynamics simulation and docking approaches. The results show that the presence of such ligands in the hydrophobic central cavity of βLG, known as the protein calyx, determines an enhancement of atomic fluctuations compared with the unliganded form, especially for loops at the entrance of the binding site. Concerted motions are evidenced for protein regions that could favor the binding of ligands. The mechanism of anchoring of fatty acids of different length is similar for the carboxylate head‐group, through electrostatic interactions with the side chains of Lys60/Lys69. The key protein residues to secure the hydrocarbon chain are Phe105/Met107, which adapt their conformation upon ligand binding. In particular, Phe105 provides an additional hydrophobic clamp only for the tail of the two fatty acids with the longest chains, palmitic, and stearic acid, which are known to bind βLG with a high affinity. The search of additional external binding sites for fatty acids, distinct from the calyx, was also carried out for palmitic acid. Two external sites with a lower affinity were identified as secondary sites, one consisting in a hydrophobic cavity allowing two distinct binding modes for the fatty acid, and the other corresponding to a surface crevice close to the protein α‐helix. The overall results provide a comprehensive picture of the dynamical behavior of βLG in complex with fatty acids, and elucidate the structural basis of the binding of these physiological ligands. Proteins 2014; 82:2609–2619. © 2014 Wiley Periodicals, Inc.     

The two coiled coils in the isolated rod domain of the intermediate filament protein desmin are staggered. A hydrodynamic analysis of tetramers and dimers

Desmin protofilaments and the proteolytically derived alpha-helical rod domain have been characterized by high-resolution gel permeation chromatography (GPC) using columns calibrated for the determination of viscosity radii. Additional characterization by chemical cross-linking and the determination of sedimentation values allowed the calculation of the molecular dimensions of the molecular species isolated. In dilute buffers GPC separated desmin rod preparations into two complexes: a dimer species (single coiled coil) with a length of 50 +/- 5 nm and a tetramer species (two coiled coils) with a length of 65 +/- 5 nm. Thus the two coiled coils in the tetramer are staggered by approximately 15 nm. The hydrodynamically derived lengths of the rod dimer and tetramer are supported by electron microscopy after metal shadowing. The hydrodynamic properties of desmin protofilaments follow that of the rod tetramer. The data on the hydrodynamic analysis of the rod tetramer of desmin in solution are in full agreement with the structural information recently deduced from paracrystals of the rod of glial fibrillary acid protein [Stewart, M., Quinlan, R.A. & Moir, R.D. (1989) J. Cell Biol. 109, 225-234]. Our results explain the inhomogeneity of molecules encountered in previous electron microscopical analyses.     

Alpha helix capping in synthetic model peptides by reciprocal side chain–main chain interactions: Evidence for an N terminal “capping box”

A significant fraction of the amino acids in proteins are alpha helical in conformation. Alpha helices in globular proteins are short, with an average length of about twelve residues, so that residues at the ends of helices make up an important fraction of all helical residues. In the middle of a helix, H-bonds connect the NH and CO groups of each residue to partners four residues along the chain. At the ends of a helix, the H-bond potential of the main chain remains unfulfilled, and helix capping interactions involving bonds from polar side chains to the NH or CO of the backbone have been proposed and detected. In a study of synthetic helical peptides, we have found that the sequence Ser-Glu-Asp-Glu stabilizes the alpha helix in a series of helical peptides with consensus sequences. Following the report by Harper and Rose, which identifies SerXaaXaaGlu as a member of a class of common motifs at the N termini of alpha helices in proteins that they refer to as “capping boxes,” we have reexamined the side chain–main chain interactions in a varient sequence using ¹H NMR, and find that the postulated reciprocal side chain-backbone bonding between the first Ser and last Glu side chains and their peptide NH partners can be resolved: Deletion of two residues N terminal to the Ser-Glu-Asp-Glu sequence in these peptides has no effect on the initiation of helical structure, as defined by two-dimensional (2D) NMR experiments on this variant. Thus the capping box sequence Ser-Glu-Asp-Glu inhibits N terminal fraying of the N terminus of alpha helix in these peptides, and shows the side chain–main chain interactions proposed by Harper and Rose. It thus acts as a helix initiating signal. Since normal a helix cannot propagate beyond the N terminus of this structure, the box acts as a termination signal in this direction as well. © 1994 John Wiley & Sons, Inc.     

Packing constraints of hydrophobic side chains in (α/β)8 barrels

An analysis of possible tight packing of hydrophobic groups simultaneously at the both surfaces of β-hyperboloid-8 was conducted. This analysis shows that the disposition of amino acid side chains at the real β-structure's surface is unique. If we sign the mean distance between adjacent β-strands as “a,” and the mean distance along β-strand between C_α atoms, whose side chains are directed to one side of the β-sheet, as “b,” the ratio b/a = √2 very precisely. This ratio ensures the most efficient packing of side hydrophobic groups at the outer surface of β-hyperboloid-8, forming, at the same time, the second by efficiency packing at its inner surface. © 1995 Wiley-Liss, Inc.     

Chemical cross-linking indicates a staggered and antiparallel protofilament of desmin intermediate filaments and characterizes one higher-level complex between protofilaments.

Tetrameric rods, protofilaments and assembled filaments of desmin, the intermediate filament protein of muscle, have been chemically cross-linked with the lysine specific cross-linkers EGS [ethylene glycol bis(succinimidylsuccinate), 1.61 nm span] and bis(sulfosuccinimidyl) suberate (1.14 nm span). One bis(sulfosuccinimidyl)suberate and two EGS cross-links were isolated from the rod and characterized. They show that the two coiled coils in the rod tetramer are staggered by approximately 15-20 nm and strongly indicate an antiparallel arrangement in which the inner overlapping part of the rod is formed by the amino-terminal helices 1A, 1B and 2A. Both EGS cross-links identified in the rod were also isolated from cross-linked filaments. The isolated rod, therefore, represents a complex also present in identical, or very similar form in protofilaments and in assembled filaments. Cross-linked filaments yielded a third EGS cross-link that must have been formed between neighboring protofilaments. It connects the highly conserved carboxy-terminus of helix 2B of the first protofilament to the overlap region formed by helices 1A and 2A of the second protofilament. The restrictions posed by these cross-links on current filament models are discussed.     

Insights into the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) by two‐dimensional Raman correlation spectroscopy

In this study, the transition process of the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) was investigated by Raman scattering and 2‐dimensional correlation spectroscopy. Temperature‐dependent Raman spectra were obtained during the helix‐sense inversion. The results of 2‐dimensional correlation analysis in the spectral regions of 1600‐1800 and 3200‐3400 cm^?1 showed that the intensity changes of the side‐chain ester C═O stretching bands occurred prior to those of amide A and amide I bands in the unwinding process of αR‐helix on heating. The sequential order of the intensity changes for amide A, amide I, and the side‐chain ester C═O stretching bands during the inversion process was determined. It was found that the conformation change of the side chain occurred prior to that of the main chain for the αR‐helix on heating. Thus, we concluded that the transformation of the backbone chain from right‐handed to left‐handed is triggered by the conformational change of the side chains.     

Effect of structural parameters of peptides on dimer formation and highly oxidized side products in the oxidation of thiols of linear analogues of human β‐defensin 3 by DMSO

The purpose of this study was to examine the effects of structural parameters of peptides on their oxidation by DMSO, including location of cysteine, effect of adjunct group participation, molecular hydrophobicity, steric hindrance or the accessibility of thiol group and peptide conformation, on oxidation rates, dimer formation and associated side products. We designed and synthesized two series of linear cysteine‐containing analogues of human β‐defensin 3 (the C1‐peptides with cysteine at the N‐terminus residue 1, the C29‐peptides with cysteine located at residue 29 in the centre of peptide), which were used for preparation of disulphide‐linked homodimers. HPLC–ESI–MS was used to monitor the oxidation process and to characterize the molecular weights of dimers and side products of high oxidation. The formations of dimers and side products were dependent on the position of cysteines. Hydrophobicity generally rendered the thiol groups less accessible and hence exposed them to slow oxidation to form dimers (or even fail to form dimers during the timescale of observation). Molecular dynamics simulations showed that the exposure of cysteines (and sulphurs) of the C1‐peptides was much larger than for the C29‐peptides. The larger hydrophobic side chains tended to enable clustering of the side chains that sequester cysteine, particularly in the C29‐peptides, which provided a molecular explanation for the observed trends in oxidation rates. Together with molecular modelling, we propose a reaction mechanism to elucidate the oxidation results of these peptides. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Stabilizing nonpolar/polar side-chain interactions in the alpha-helix.

A simplistic, yet often used, view of protein stability is that amino acids attract other amino acids with similar polarity, whereas nonpolar and polar side chains repel. Here we show that nonpolar/polar interactions, namely Val or Ile bonding to Lys or Arg in alpha-helices, can in fact be stabilizing. Residues spaced i, i + 4 in alpha-helices are on the same face of the helix, with potential to favorably interact and stabilize the structure. We observe that the nonpolar/polar pairs Ile-Lys, Ile-Arg, and Val-Lys occur in protein helices more often than expected when spaced i, i + 4. Partially helical peptides containing pairs of nonpolar/polar residues were synthesized. Controls with i, i + 5 spacing have the residues on opposite faces of the helix and are less helical than the test peptides with the i, i + 4 interactions. Experimental circular dichroism results were analyzed with helix-coil theory to calculate the free energy for the interactions. All three stabilize the helix with DeltaG between -0.14 and -0.32 kcal x mol(-1). The interactions are hydrophobic with contacts between Val or Ile and the alkyl groups in Arg or Lys. Side chains such as Lys and Arg can thus interact favorably with both polar and nonpolar residues.     

Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers.

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.     

Structural properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

Replica exchange molecular dynamics simulations (300 ns) were used to study the dimerization of amyloid β(1‐40) (Aβ(1‐40)) polypeptide. Configurational entropy calculations revealed that at physiological temperature (310 K, 37°C) dynamic dimers are formed by randomly docked monomers. Free energy of binding of the two chains to each other was ?93.56 ± 6.341 kJ mol^?1. Prevalence of random coil conformations was found for both chains with the exceptions of increased β‐sheet content from residues 16‐21 and 29‐32 of chain A and residues 15‐21 and 30‐33 of chain B with β‐turn/β‐bend conformations in both chains from residues 1‐16, 21‐29 of chain A, 1‐16, and 21‐29 of chain B. There is a mixed β‐turn/β‐sheet region from residues 33‐38 of both chains. Analysis of intra‐ and interchain residue distances shows that, although the individual chains are highly flexible, the dimer system stays in a loosely packed antiparallel β‐sheet configuration with contacts between residues 17‐21 of chain A with residues 17‐21 and 31‐36 of chain B as well as residues 31‐36 of chain A with residues 17‐21 and 31‐36 of chain B. Based on dihedral principal component analysis, the antiparallel β‐sheet‐loop‐β‐sheet conformational motif is favored for many low energy sampled conformations. Our results show that Aβ(1‐40) can form dynamic dimers in aqueous solution that have significant conformational flexibility and are stabilized by collapse of the central and C‐terminal hydrophobic cores with the expected β‐sheet‐loop‐β‐sheet conformational motif. Proteins 2017; 85:1024–1045. © 2017 Wiley Periodicals, Inc.     

Effect of the side group on the helix-forming tendency of α-alkyl-β-L-aspartamyl residues

The conformational preferences of the monomeric units of a series of poly(α-alkyl-β-L-aspartate)s were examined by quantum mechanical calculations. α-Alkyl-β-aspartamyl m-oligopeptides with a number of residues m ranging from 1 to 7 and arranged in the conformations experimentally observed for their corresponding polymers were computed. Both their total relative energies and their cooperative energy differences were compared as a function of the length of the oligopeptide and the nature of the alkyl side group. Results revealed that the 13/4 helical arrangement is the most stable structure for the isolated polymer chain for any side group, although a 17/4 helix becomes favored in the case of methyl and ethyl groups due to the packing effects. On the other hand, the stability of the 4/1 helix appears to be the preferred conformation for side groups with a branched constitution. © 1997 John Wiley & Sons, Inc. Biopoly 41: 721–729, 1997     

Helix-forming tendencies of nonpolar amino acids predicted by Monte Carlo simulated annealing

Monte Carlo simulated annealing is applied to the study of the α-helix-forming tendencies of seven nonpolar amino acids, Ala, Leu, Met, Phe, Ile, Val, and Gly. Homooligomers of 10 amino acids are used and the helix tendency is calculated by folding α-helicies from completely random initial conformations. The results of the simulation imply that Met, Ala, and Leu are helix formers and that Val, Ile, and Gly are helix breakers, while Phe comes in between the two groups. The differences between helix formers and breakers turned out to be large in agreement with the recent experiments with short peptides. It is argued from the energy distributions of the obtained conformations that the helix tendency is small for the helix breakers because of steric hindrance of side chains. Homoglycine is shown to favor a random coil conformation. The β-strand tendencies of the same homooligomers are also considered, and they are shown to agree with the frequencies of amino acids in β-sheet from the protein data base. © 1994 Wiley-Liss, Inc.     

3(10)-helices in proteins are parahelices

The 3(10)-helix is characterized by having at least two consecutive hydrogen bonds between the main-chain carbonyl oxygen of residue i and the main-chain amide hydrogen of residue i + 3. The helical parameters--pitch, residues per turn, radius, and root mean square deviation (rmsd) from the best-fit helix--were determined by using the HELFIT program. All 3(10)-helices were classified as regular or irregular based on rmsd/(N - 1)1/2 where N is the helix length. For both there are systematic, position-specific shifts in the backbone dihedral angles. The average phi, psi shift systematically from approximately -58 degrees, approximately -32 degrees to approximately -90 degrees, approximately -4 degrees for helices 5, 6, and 7 residues long. The same general pattern is seen for helices, N = 8 and 9; however, in N = 9, the trend is repeated with residues 6, 7, and 8 approximately repeating the phi, psi of residues 2, 3, and 4. The residues per turn and radius of regular 3(10)-helices decrease with increasing length of helix, while the helix pitch and rise per residue increase. That is, regular 3(10)-helices become thinner and longer as N increases from 5 to 8. The fraction of regular 3(10)-helices decreases linearly with helix length. All longer helices, N > or = 9 are irregular. Energy minimizations show that regular helices become less stable with increasing helix length. These findings indicate that the definition of 3(10)-helices in terms of average, uniform dihedral angles is not appropriate and that it is inherently unstable for a polypeptide to form an extended, regular 3(10)-helix. The 3(10)-helices observed in proteins are better referred to parahelices.     

The structure of melittin in the form I crystals and its implication for melittin's lytic and surface activities.

Melittin from bee venom is water-soluble, yet integrates into membranes and lyses cells. Each melittin chain consists of 26 amino acid residues and in aqueous salt solutions it exists as a tetramer. We have determined the molecular structure of the tetramer in two crystal forms grown from concentrated salt solutions. In both crystal forms the melittin polypeptide is a bent alpha-helical rod, with the "inner" surface largely consisting of hydrophobic sidechains and the "outer" surface consisting of hydrophilic side chains. Thus, the helix is strongly amphiphilic. In the tetramer, four such helices contribute their hydrophobic side chains to the center of the molecule. The packing of melittin tetramers is also very similar in the two crystal forms: they are packed in planar layers with the outsides forming hydrophilic surfaces and the insides (the centers of melittin tetramers) forming a hydrophobic surface. We suggest that the surface activity of melittin can be rationalized in terms of these surfaces. The lytic activity of melittin can also be interpreted in terms of the molecular structure observed in the crystals: the hydrophobic inner surface of a melittin helix may integrate into the apolar region of a bilayer with the helix axis approximately parallel to the plane of the bilayer, and with the hydrophilic surface exposed to the aqueous phase. This integration would be expected to disrupt the bilayer because of melittin helix would penetrate only a short distance into it. Additionally, the integration of melittin from one side of a bilayer would produce a surface area difference across the bilayer, perhaps leading to lysis. In this view, melittin is distinct from membrane proteins that penetrate evenly into both leaflets of a bilayer or exactly halfway through a bilayer, and hence we refer to melittin as a surface-active protein.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.