Harnessing the Unique Structural Properties of Isolated α-Helices

Abstract

The α-helix is a ubiquitous secondary structural element that is almost exclusively observed in proteins when stabilized by tertiary or quaternary interactions. However, beginning with the unexpected observations of α-helix formation in the isolated C-peptide in ribonuclease A, there is growing evidence that a significant percentage (0.2%) of all proteins contain isolated stable single α-helical domains (SAH). These SAH domains provide unique structural features essential for normal protein function. A subset of SAH domains contain a characteristic ER/K motif, composed of a repeating sequence of ∼4 consecutive glutamic acids followed by ∼4 consecutive basic arginine or lysine (R/K) residues. The ER/K α-helix, also termed the ER/K linker, has been extensively characterized in the context of the myosin family of molecular motors and is emerging as a versatile structural element for protein and cellular engineering applications. Here, we review the structure and function of SAH domains, as well as the tools to identify them in natural proteins. We conclude with a discussion of recent studies that have successfully used the modular ER/K linker for engineering chimeric myosin proteins with altered mechanical properties, as well as synthetic polypeptides that can be used to monitor and systematically modulate protein interactions within cells. The α-helix is a ubiquitous secondary structural element that is almost exclusively observed in proteins when stabilized by tertiary or quaternary interactions. However, beginning with the unexpected observations of α-helix formation in the isolated C-peptide in ribonuclease A, there is growing evidence that a significant percentage (0.2%) of all proteins contain isolated stable single α-helical domains (SAH). These SAH domains provide unique structural features essential for normal protein function. A subset of SAH domains contain a characteristic ER/K motif, composed of a repeating sequence of ∼4 consecutive glutamic acids followed by ∼4 consecutive basic arginine or lysine (R/K) residues. The ER/K α-helix, also termed the ER/K linker, has been extensively characterized in the context of the myosin family of molecular motors and is emerging as a versatile structural element for protein and cellular engineering applications. Here, we review the structure and function of SAH domains, as well as the tools to identify them in natural proteins. We conclude with a discussion of recent studies that have successfully used the modular ER/K linker for engineering chimeric myosin proteins with altered mechanical properties, as well as synthetic polypeptides that can be used to monitor and systematically modulate protein interactions within cells. Until recently, SAH 2The abbreviations used are: SAHsingle α-helical domainCSAHcharged SAHEBFPenhanced blue fluorescent proteinEGFPenhanced green fluorescent proteinSAXSsmall angle x-ray scatteringMDmolecular dynamicsPDCD5programmed cell death 5CaMcalmodulinSPASMSystematic Protein Affinity Strength ModulationFAKfocal adhesion kinaseMyomyosin. domains in natural proteins were predicted by secondary structure prediction algorithms to form a coiled-coil, in part due to the high concentration of charged and polar residues that are also the hallmark of the coiled-coil motif (1.Knight P.J. Thirumurugan K. Xu Y. Wang F. Kalverda A.P. Stafford 3rd, W.F. Sellers J.R. Peckham M. The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head.J. Biol. Chem. 2005; 280: 34702-34708Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Among the multiple folds in globular proteins that stabilize α-helices, the coiled-coil motif has been extensively characterized and in general is the most predictable form of tertiary protein structure (2.Woolfson D.N. The design of coiled-coil structures and assemblies.Adv. Protein Chem. 2005; 70: 79-112Crossref PubMed Scopus (474) Google Scholar). In the coiled-coil motif, two or more α-helices are individually stabilized by sequence-specific packing at consensus hydrophobic patches. Extensive studies have elicited general sequence and structure rules that govern coiled-coil interactions. Briefly, the amino acid sequence of each α-helix in a coiled-coil is divided into heptads (7 residues) that form nearly two complete α-helical turns and span 1.05 nm along the helical axis. Each amino acid in the heptad is described by its relative position, moving from the N to C terminus, using the nomenclature abcdefg. In canonical dimeric coiled-coils, the a and d positions radiate away from the core of the α-helix, 60° apart and offset by 0.45 nm along the helix length, and are typically occupied by aliphatic hydrophobic residues, whereas polar residues comprise the rest of the positions. As the heptad is repeated, it forms a continuous hydrophobic patch located along a single face of the α-helix compatible with a tight intermolecular interaction between polypeptides with the same or similar heptad pattern (2.Woolfson D.N. The design of coiled-coil structures and assemblies.Adv. Protein Chem. 2005; 70: 79-112Crossref PubMed Scopus (474) Google Scholar). However, often polar or charged residues occupy the a and d positions, leading to local destabilization of the coiled-coil motif (2.Woolfson D.N. The design of coiled-coil structures and assemblies.Adv. Protein Chem. 2005; 70: 79-112Crossref PubMed Scopus (474) Google Scholar, 3.Sunitha M.S. Nair A.G. Charya A. Jadhav K. Mukhopadhyay S. Sowdhamini R. Structural attributes for the recognition of weak and anomalous regions in coiled-coils of myosins and other motor proteins.BMC Res. Notes. 2012; 5: 530Crossref PubMed Scopus (12) Google Scholar, 4.Kreuzer S.M. Elber R. Coiled-coil response to mechanical force: global stability and local cracking.Biophys. J. 2013; 105: 951-961Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). By extension, when most or all of the a and d sites in consecutive heptads are occupied by polar residues, individual α-helices cannot be mutually stabilized through hydrophobic packing. In this event, the polypeptide either will exist as a monomeric random coil or in some cases will form an SAH domain. The SAH domain is a stable, monomeric, extended α-helix that is encoded by its primary amino acid sequence and exists in polar solvent independent of tertiary interactions with other protein motifs. single α-helical domain charged SAH enhanced blue fluorescent protein enhanced green fluorescent protein small angle x-ray scattering molecular dynamics programmed cell death 5 calmodulin Systematic Protein Affinity Strength Modulation focal adhesion kinase myosin. The rules governing the helicity of isolated peptides have been extensively studied. We highlight select studies of particular interest, and refer to Ref. 5.Scholtz J.M. Baldwin R.L. The mechanism of α-helix formation by peptides.Annu. Rev. Biophys. Biomol. Struct. 1992; 21: 95-118Crossref PubMed Scopus (480) Google Scholar for a more comprehensive review. Prior to work on synthetic peptides from the Baldwin laboratory, including one derived from ribonuclease A (6.Marqusee S. Robbins V.H. Baldwin R.L. Unusually stable helix formation in short alanine-based peptides.Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 5286-5290Crossref PubMed Scopus (732) Google Scholar, 7.Marqusee S. Baldwin R.L. Helix stabilization by Glu−…Lys+ salt bridges in short peptides of de novo design.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 8898-8902Crossref PubMed Scopus (900) Google Scholar, 8.Bierzynski A. Kim P.S. Baldwin R.L. A salt bridge stabilizes the helix formed by isolated C-peptide of RNase A.Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 2470-2474Crossref PubMed Scopus (276) Google Scholar), peptides shorter than 20 amino acids were not expected to show measurable helix formation in aqueous solution based on the statistical Zimm-Bragg model, which focuses on the propagation of spontaneous helicity and does not account for long distance electrostatic interactions between side chains (9.Zimm B.H. Bragg J.K. Theory of the phase transition between helix and random coil in polypeptide chains.J. Chem. Phys. 1959; 31: 526-535Crossref Scopus (1676) Google Scholar). Marqusee et al. (6.Marqusee S. Robbins V.H. Baldwin R.L. Unusually stable helix formation in short alanine-based peptides.Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 5286-5290Crossref PubMed Scopus (732) Google Scholar) found that a synthetic peptide, 16 residues long, containing primarily alanine residues sparsely interspersed with a single Glu or Lys for solubilization, had high helical content (up to 80%) in aqueous solution as measured by CD. This study highlighted the inherent helix-forming potential of alanine in the absence of electrostatic interactions between side chains. In a separate study, Marqusee and Baldwin (7.Marqusee S. Baldwin R.L. Helix stabilization by Glu−…Lys+ salt bridges in short peptides of de novo design.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 8898-8902Crossref PubMed Scopus (900) Google Scholar) synthesized peptides containing 16 residues with three pairs of a Glu and a Lys separated by either 3 (i, i + 3) or 4 (i, i + 4) alanines. These peptides were synthesized, in part, to characterize the influence of electrostatic interactions between Glu and Lys on α-helix formation. Both peptides were soluble and monomeric in aqueous solution and had detectable helical content as determined by CD. However, the (i, i + 4) (i.e. (EAAAK)3) spacing yielded significantly higher helicity when compared with (i, i + 3), presumably due to the preferred rotamer configurations of the Glu and Lys side chains. Interestingly, the measured helicity was preserved at extremes of pH (from 2 to 12) and at high concentrations of NaCl, suggesting that the electrostatic interactions were primarily derived from salt bridges (H-bonded ionic interactions) rather than direct ionic interactions. The helix-stabilizing effects of E-K interactions on an alanine backbone were subsequently extended to D-K, E-R, and D-R pairs (10.Huyghues-Despointes B.M. Scholtz J.M. Baldwin R.L. Helical peptides with three pairs of Asp-Arg and Glu-Arg residues in different orientations and spacings.Protein Sci. 1993; 2: 80-85Crossref PubMed Scopus (126) Google Scholar). A subsequent study with alanine-based peptides also defined the role of capping residues at the N and C termini in stabilizing isolated α-helices. In general, α-helix formation is aided by negatively charged residues at the N terminus and positively charged residues at the C terminus (11.Doig A.J. Baldwin R.L. N- and C-capping preferences for all 20 amino acids in α-helical peptides.Protein Sci. 1995; 4: 1325-1336Crossref PubMed Scopus Google Scholar). This is with an interaction of charged capping residues with the of the protein α-helix, which is from its C to N in the context of the charged residues at the can also stabilizing electrostatic interactions with the and (11.Doig A.J. Baldwin R.L. N- and C-capping preferences for all 20 amino acids in α-helical peptides.Protein Sci. 1995; 4: 1325-1336Crossref PubMed Scopus Google Scholar). These studies of synthetic alanine-based peptides and the for and the stability of isolated α-helices. The motif was subsequently into et al. R. A. of the which separate domains of a PubMed Scopus Google Scholar) the of as spacing between a of green fluorescent protein and The not with and as from between and whereas helicity with linker length, suggesting that stabilization of the α-helix the spacing between the fluorescent proteins to the of the In a subsequent study, the of the were by small angle x-ray scattering 3) whereas 4) monomeric at high concentrations The of with linker and is higher than of the same R. Y. of chimeric proteins by PubMed Scopus Google Scholar). studies the of the as extended between this structural the linker has been to of the helical linker to of Res. PubMed Scopus Google Scholar) and Y. the of and protein by Res. PubMed Scopus Google Scholar) of proteins. However, of for the of peptides with this particular linker, the motif has been to have at pH Protein a unique of a Sci. 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Stafford W.F. A long helix from the of Biol. Chem. Full Text PDF PubMed Google Scholar) extensively characterized a of that this ER/K which is with a continuous acid α-helical The of this polypeptide in a monomeric a of protein concentrations with an of nm that is the predicted of an extended The in was significantly than coiled-coils from or the myosin suggesting a single extended In to stabilizing the α-helix, Wang et al. J.M. K. Stafford W.F. A long helix from the of Biol. Chem. Full Text PDF PubMed Google Scholar) that the salt bridges this α-helix from with and myosin through domains located at the of its SAH domain. to the of it does not the interaction Sellers J.R. The effects of on and of to Biol. Chem. Full Text PDF PubMed Google Scholar). the extended single α-helix in as a to this However, not all SAH domains are expected to function as for the programmed cell death 5 The acid of was by et al. Y. Y. Y. Wang J. The of programmed cell death 5 protein can form a stable α-helix unique electrostatic potential J. 2005; PubMed Scopus Google Scholar) to form a single α-helix by and of the α-helix of significantly the effects by on the of and et al. Y. Y. Y. Wang J. The of programmed cell death 5 protein can form a stable α-helix unique electrostatic potential J. 2005; PubMed Scopus Google Scholar) a role for this SAH domain in the of The high structure of an SAH domain was in a structure of the protein structure of protein a J. PubMed Google Scholar). This protein a and extended acid linker between two globular domains A subsequent study synthesized the and found that the was primarily monomeric at concentrations to in studies and primarily α-helical as determined by a higher of helicity than was expected based on its primary sequence R. stable helix from the protein for protein and Biol. PubMed Scopus Google Scholar). found that the helicity of the be by salt concentrations of ionic side interactions to the stability of the this is from the motif, it does have predicted (i, i + 4) electrostatic interactions. The single α-helix was to as a between two globular domains of the protein that are to In to its structural role in the the also that the of the α-helix and stabilize the of the two domains at its the α-helix does the the of the two domains at the will be The most extensively characterized SAH domains to are the ER/K α-helices found in myosin and A study by et al. (1.Knight P.J. Thirumurugan K. Xu Y. Wang F. Kalverda A.P. Stafford 3rd, W.F. Sellers J.R. Peckham M. The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head.J. Biol. Chem. 2005; 280: 34702-34708Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) a coiled-coil motif in myosin that was in charged residues in the a and d positions of the heptad The this as a that not with coiled-coil and the SAH to an isolated stable single α-helix The an ER/K found that the residues form a random whereas all other residues in the are determined the to be monomeric at concentrations to found that the α-helical as determined by is to higher salt concentrations than a synthetic acid peptide, presumably due to more stable electrostatic interactions. The study also the SAH domain in the context of a nearly myosin which a coiled-coil amino acids by and found that of the was monomeric, whereas in the dimeric a small of the α-helical to be The of the myosin was nm than expected nm an expected to the expected of nm the acid were in an extended this study for two SAH domains in regions described as coiled-coil regions in myosin and based on the ER/K motif in primary amino acid of which were subsequently et al. S. J. S. single α-helical domains bridge the between structure and function of myosin Struct. Biol. PubMed Scopus Google Scholar) that an ER/K motif was for the of dimeric myosin The myosin was to form a coiled-coil S. A domain is essential for the and of myosin 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) based on a prediction of the et al. S. J. S. single α-helical domains bridge the between structure and function of myosin Struct. Biol. PubMed Scopus Google Scholar) that a polypeptide derived from the to a characteristic of coiled-coil a of the was found to be monomeric at high concentrations an extended with the as an ER/K motif of the by of a canonical coiled-coil at its N terminus, the and of myosin The of this ER/K α-helix is in its to the mechanical of myosin as it an by S. J. M. S. and x-ray scattering to the of a protein ER/K J. Full Text Full Text PDF PubMed Scopus Google Scholar). By and of this and ER/K motif and the of the ER/K motif based on an was to be nm S. J. M. S. and x-ray scattering to the of a protein ER/K J. Full Text Full Text PDF PubMed Scopus Google Scholar) Until recently, the of the SAH domain was to the of natural proteins it had been for the ER/K motif et al. S. S. interactions in the ER/K α-helical protein Natl. Acad. Sci. U.S.A. 105: PubMed Scopus Google Scholar) which had an of to this motif, in from to Peckham et al. M. P.J. a predicted coil is a single in myosins and other 5: Scholar) to identify SAH domains by predicted to be coiled-coils based on high that to have hydrophobic residues in the a and d a of coiled-coil domains that were to the SAH domain in myosin found that to of proteins as coiled-coils be SAH domains This study an of of all proteins in the that contain an SAH domain M. P.J. a predicted coil is a single in myosins and other 5: Scholar). et al. single a versatile protein structural PubMed Scopus Google Scholar) a more and to identify charged SAH two different one a function that characteristic (i, i + 4) or (i, i + 3) salt bridges and the one a that residues heptad apart These SAH domains by the for regions than amino acids single a versatile protein structural PubMed Scopus Google Scholar). Both are at the charged SAH to the provide a of of all proteins in an as containing structural Interestingly, were to have the of in almost proteins in this one had a high of the of x-ray structures of The had high with and coiled-coil motifs. The that this motif be and that single to of between coiled-coil, and A. single α-helices in by a consensus prediction Biophys. 2012; PubMed Scopus Google Scholar). As an to between SAH and coiled-coil domains, et al. M.S. Nair A.G. Charya A. Jadhav K. Mukhopadhyay S. Sowdhamini R. Structural attributes for the recognition of weak and anomalous regions in coiled-coils of myosins and other motor proteins.BMC Res. Notes. 2012; 5: 530Crossref PubMed Scopus (12) Google Scholar) have a termed This the of the of a potential coiled-coil interaction at the based on the relative of the charged residues and the characteristic pattern of hydrophobic residues. it that SAH domains are The ER/K motif has found in protein separate studies have used chimeric to the of ER/K α-helix mechanical on myosin function et al. S.M. Y. P.J. Sellers J.R. Peckham M. The SAH domain the of the myosin Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) that an ER/K α-helix can function as a in myosin by a single myosin nearly as as its However, in to the the ER/K α-helix was not to the of the two myosin within a single due to its and S. domain of myosin for Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the ER/K α-helix from myosin was and to of myosin and myosin on into the myosin for suggesting that the ER/K α-helix the of the myosin within a et al. M. S. on cellular Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) found that of myosin not myosin on a cellular that myosin was to the of on cellular a of the myosin with the ER/K α-helix from myosin was to myosin and studies bridge the mechanical of ER/K α-helices with in myosin. In a different to protein the ER/K linker has also been used to and the concentration of interactions and S. Systematic of protein interaction using a modular ER/K α-helix Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) a single polypeptide containing an ER/K linker with an calmodulin and by a linker and a with a linker to a to with the The interaction between and its was by in between and In the absence of significant was with ER/K of amino acids or more to nm in along the α-helical A in was observed the of was independent of the concentration of at the that an interaction was the on either of the ER/K linker into of with concentrations of was used to the concentration of the of the the concentration by an of for each 10 nm of ER/K ER/K linker from 10 to nm altered the concentration of the interaction from 10 to nm the concentration of a linker is the stability of proteins by linker and Natl. Acad. Sci. U.S.A. PubMed Scopus Google similar are observed when and the are by different peptides This is in to the expected of an is that a in the of the ER/K linker, which the of the However, the measured was found to be independent of ER/K linker The a structural of observations by suggesting that the ER/K linker in helicity that to interactions between the The of with α-helix However, the are to be in that the in to interactions. this it is with a study suggesting that α-helical peptides along S. is an of the of of helical peptides by scattering and molecular Biol. 2005; PubMed Scopus Google Scholar). The of the ER/K linker to modulate interactions was termed Systematic Protein Affinity Strength Modulation A of the is its and the individual domains can be with basic molecular tools in most The has been used to interactions in cells. polypeptide based on have been used to of M. S. of in Biol. Chem. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar) concentration with ER/K linker has been used to the of focal adhesion kinase and the effects of kinase and interactions in cellular M. S. and focal adhesion kinase in Biol. Chem. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar) The ER/K linker the of with in the absence of an This has been used to the pH of for the effects of pH on the of the ER/K linker has found in interactions in which has yielded a of and intermolecular interactions in protein kinase C M. Wang A. M. S. modular domains to Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) we that the ER/K linker be used in with other synthetic protein including proteins and protein as a to study individual interactions or to cellular The SAH domain is a structural element found in it to as a structural element that globular it has been extensively characterized in a natural its sequence and structural for its from The ER/K motif is a subset of SAH domains that have been extensively characterized through studies of the myosin family of molecular ER/K α-helices, encoded by this motif, have found for or systematically protein interactions. The of the ER/K motif it a versatile protein structural element in an of that are to an cellular In to cellular on an ER/K have direct for small