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Helical assemblies: Structure determinants Natalya A. Kurochkina a,n, Michael J. Iadarola b,1 a
The School of Theoretical Modeling, 1629 K St NW s 300, Washington, DC 20006, United States Anesthesia Section, Department of Periorative Medicine, Clinical Center, NIH, Building 10, Room 2C401, 10 Center Drive, MSC 1510, Bethesda, MD 20892, United States
b
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
Pocket geometry of α-helix contributes to chirality and curvature of the helical assembly. Orientation of helical edges determines direction of the assembly and influences curvature of the assembly. Amino acid composition of α-helix determines its pocket geometry.
art ic l e i nf o
a b s t r a c t
Article history: Received 22 September 2014 Received in revised form 12 January 2015 Accepted 14 January 2015
Protein structural motifs such as helical assemblies and α⧸β barrels combine secondary structure elements with various types of interactions. Helix–helix interfaces of assemblies – Ankyrin, ARM/HEAT, PUM, LRR, and TPR repeats – exhibit unique amino acid composition and patterns of interactions that correlate with curvature of solenoids, surface geometry and mutual orientation of the helical edges. Inner rows of ankyrin, ARM/HEAT, and PUM-HD repeats utilize edges (i 1, i) and (iþ 1, i þ2) for the interaction of the given α-helix with preceding and following helices correspondingly, whereas outer rows of these proteins and LRR repeats invert this pattern and utilize edges (i 1, i) and (i 3, i 2). Arrangement of contacts observed in protein ligands that bind helical assemblies has to mimic the assembly pattern to provide the same curvature as a determinant of binding specificity. These characteristics are important for understanding fold recognition, specificity of protein–protein interactions, and design of new drugs and materials. & 2015 Published by Elsevier Ltd.
Keywords: Protein conformation Repeats Helix–helix interface Assembly Enantiomer-selective ligand binding
1. Introduction Helical assemblies are essential modules of many cellular processes that regulate events of transcription, sensory transduction, development, recognition, and communication (Andrade et al., 2001; deWit et al., 2011; Blatch and Lassle, 1999; Sawyer et al., 2013). As mediators of protein–protein interactions and cell signaling, helical structures provide a basis for our understanding n
Corresponding author. Tel.: þ 1 240 381 2383. E-mail addresses: [email protected] (N.A. Kurochkina), [email protected] (M.J. Iadarola). 1 Tel.: þ 1 301 496 2758.
of many pathologies such as mental, degenerative and immune system diseases, cancer, and inflammation (Utreras et al., 2013; Lishko et al., 2007; Sanders et al., 2014; Holzer and Izzo, 2014; Latorre et al., 2009). Many helix bundles have been shown to be vital for the development of drugs against influenza, obesity/ diabetes, hepatitis C virus and other diseases (Schnell and Chou, 2008; Berardi et al., 2011; OuYang et al., 2013). Designed repeat proteins have the ability to bind their specific targets and provide drug candidates for future treatments (Stumpp et al., 2008; Abil et al., 2014). Structure of the helical assembly can contain one or more rows of stacked helices that form a solenoid, a helix of helices. Each type of the repeat – Ankyrin, ARM/HEAT, Pumilio homology domain (PUM-HD), leucine rich repeat (LRR), or
http://dx.doi.org/10.1016/j.jtbi.2015.01.012 0022-5193/& 2015 Published by Elsevier Ltd.
Please cite this article as: Kurochkina, N.A., Iadarola, M.J., Helical assemblies: Structure determinants. J. Theor. Biol. (2015), http://dx.doi. org/10.1016/j.jtbi.2015.01.012i
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tetratricopeptide (TPR) – exhibits unique properties, characteristic amino acid composition (Andrade et al., 2001; deWit et al., 2011; Blatch and Lassle, 1999; Sawyer et al., 2013), and groups of consensus sequences (Mosavi et al., 2002; Gaudet, 2008). The structure of the α-helix organizes the protein backbone in a specific hydrogen bonding pattern (Pauling et al., 1951). Arrangements and energetics of interactions of α-helices, β-sheets, and loops have been extensively studied with model peptides (Chou et al., 1988, 1989, 1990a, 1990b) and proteins including 8α/8β barrels (Chou and Carlacci, 1991), α-helix bundles (Carlacci, 1990b, 1990c, 1991; Carlacci and Maggiora., 1991; Chou et al., 1992a, 1992b), leucine zippers (Chou, 1992), and globins (Gerritsen et al., 1985). Packing of secondary structure elements “knobs into holes” (Crick, 1953), complementarity of interacting surfaces (Chothia et al., 1981), hydrogen bonding and van der Waals interactions contribute to specificity and stability of protein molecules and energy of hydrophobic and electrostatic interactions (Scheraga et al., 1982; Schulz and Schirmer, 1982; Chou et al., 1983, 1984; Carlacci, 1990a, 1991). Formation of pathogenic β-sheet aggregates from α-helix prion proteins demonstrates polypeptide chain conformational transitions under various conditions (Zhou, 2011b; Zhou and Huang, 2013). The hydrophobic and hydrophilic environment of α-helices is one of the major factors influencing their structural properties (Chou et al., 1997). The distribution of amino acids at the helical surfaces of leucine zipper dimeric molecular complexes clearly shows clustering of hydrophobic residues at interface positions a and d and hydrophilic residues at the interface with solvent (Chou et al., 1990a, 1997, 2011; Zhou, 2011a). As an α-helix binds more ligands, hydrophobic patches expand so that adjacent edges become involved in helix–helix interactions. In membrane helices or helices surrounded by other secondary structure elements, all edges are hydrophobic since they are not exposed to polar solvent. Hydrophobic interactions determined by specific amino acid combinations are important structural determinants of these oligomers. Amino acid combinations characteristic for each type of helix– helix interface and arrangement of α-helices in proteins show good correlation (Kurochkina, 2008; Kurochkina and Choekyi, 2011). Specific combinations at particular helical edges are important for the shape of the assembly as was previously shown for 8α/ 8β TIM-barrel proteins and 4-α-helix subunits of tobacco mosaic virus (Kurochkina, 2010). In the present work, we demonstrate that arrangement of amino acids at the helical edges and specific amino acid combinations of helix–helix interfaces can distinguish one type of helical assembly from another. Inner rows of ankyrin, ARM/HEAT, and PUM-HD repeats utilize edges (i 1, i) and (iþ1, iþ2) for the interaction of the given α-helix with preceding and following helices, respectively, whereas outer rows of these proteins and LRR repeats invert this pattern and utilize edges (i 1, i) and (i 3, i 2). The reason that this inversion of contacts leads to the change in handedness of the assembly can be explained by the geometry of the helical surface and mutual orientation of the helical edges. Each of the two different contact patterns corresponds to a unique helix arrangement. The new approach elaborated can be used to address mechanisms of action of protein molecules, prediction of specific protein–protein interactions, fold recognition, and design of drugs, nanostructures and nanomaterials.
containing peptide groups of hydrogen-bonded residues. A helical edge that contains Cα atoms of the two consecutive amino acids together with all atoms located between α-carbons forms such a plane. An edge can be designated by two consecutive Cα atoms, for example, (i, iþ1) or (iþ1, i þ2) (Fig. 1A). Edges and peptide planes are important for determining both the α-helix shape and recognition of binding surfaces by secondary structure elements. The helix–helix interface is formed by amino acids located mainly at the conserved core positions a and d and less conserved but more exposed positions e and g in leucine zipper nomenclature (Kohn et al., 1977). Each type of the repeats assembly has a repeat unit: a pair of α-helices and a β-hairpin (ankyrin), two (HEAT, TPR) or three (ARM) α-helices, or α-helix and β-strand (LRR) stacked so that they form one or two rows of α-helices (Fig. 1B and C). In the ankyrin repeat molecules, each inner row helix (A) forms an antiparallel interface with outer row helix (B) and two parallel interfaces, one with preceding (A0 ) and one with following (A00 ) helices. As a result, AB, AA0 , and AA00 interfaces are observed in the inner row, and BB0 and BB00 interfaces in the outer row (Fig. 1C). A similar arrangement of helices is present in PUM and ARM/HEAT repeats but they differ in the number of repeats per helical turn, interhelical angles, and structure of the repeat unit. In ARM/HEAT repeats, B helices form an inner (concave) row whereas A helices form an outer (convex) row. In the LRR repeat unit, outer row helices wrap around an inner row β-sheet and a second row of helices (Fig. 1B). Although outer rows of ankyrin, ARM/HEAT, PUM, and LRR repeats have a similar organization (Fig. 1B and C), the direction of the assembly is opposite to that of inner rows. These two types of assemblies cannot be superimposed. The difference in the outline of each assembly can be clearly seen if positions a of the row helices of the two assembly types are shown in the same coordinate system (Fig. 1D). This coordinate system is selected so that the α-carbon of amino acid at position a is at the origin, the peptide group between residue at position a and residue at position g preceding a is in XZ plane, vector from Cα at position g to Cβ at position a is parallel to the X axis, and the negative end of the Y-axis points toward the interacting helix (Kurochkina, 2008). Coordinates of each row of helices are transformed so that position a of the N-terminal helix interface with the following helix is at the origin, and peptide group of the residues at positions g and a is in XZ plane. All consecutive helices of the row will follow in the negative Y direction. We can see that inner and outer rows follow opposite X axis directions. Assignment of positions a, d, e, and g to each helix–helix interface (Fig. 1E) reveals that contacts of the central helix with three surrounding helices follow a particular pattern that is repeated at each ankyrin unit. This pattern differs from the pattern of other helix–helix interfaces. For instance, parallel interfaces of the TIMbarrel proteins utilize edges (i, iþ1) and (iþ5, iþ6) to contact the preceding and following helices (Kurochkina, 2010), whereas the inner rows of ankyrin repeats use (i 1, i) and (iþ 1, iþ 2). This same pattern of contacts is observed at the helix–helix interfaces of the inner rows of ARM/HEAT and PUM-HD assemblies. However, in the outer rows of these proteins and LRR repeats, the pattern of contacts is inverted: edges (i1, i) and (i 3, i 2) are involved in contacts of the central helix with the preceding and following helices. How does this inversion result in the change of the direction of the solenoid producing two types of assemblies that cannot be superimposed?
2. Results
2.2. Pocket geometry and chirality
2.1. Solenoid structures and patterns of interactions
If we draw a plane perpendicular to the peptide group plane of the residues (i 1, i), we can see that the edges (i 3, i 2) and (iþ1, iþ 2) are mirror images of each other (Fig. 1A). Conserved feature of any α-helix is that angle of the edge (i 3, i 2) with the peptide
Each peptide group comprises two amino acids joined by a peptide bond. The surface of an α-helix is shaped by planes
Please cite this article as: Kurochkina, N.A., Iadarola, M.J., Helical assemblies: Structure determinants. J. Theor. Biol. (2015), http://dx.doi. org/10.1016/j.jtbi.2015.01.012i
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Fig. 1. Helical assemblies. (A) Helical wheel. Interactions of the given helix (A) with preceding (A0 0 ) and following (A0 ) helices involve edges (i 1, i; □) and (iþ 1, iþ 2) in the ankyrin inner row ( ), PUM ( ), and ARM/HEAT ( ) repeats but (i 1, i; □) and (i 3, i 2) in LRR repeats ( ) and ankyrin outer row ( ). Leading edges are designated by filled boxes. Edges (i 3, i 2) and (i þ1, iþ 2) are nonsuperimposable mirror images of each other. (B) LRR repeats of Internalin A (INLA). (C) Ankyrin repeats of ankyrinR inner row. (D) The line connecting positions a of the repeats in the XY plane. The lowest and highest points in the Z direction are shown by empty and filled enlarged markers correspondingly. (E) Comparison of characteristic pattern of contacts in ankyrinR repeats and internalin A LRR repeats. Amino acid sequence alignment of the repeats and their consensus sequences; α-helices are boxed; ψ–hydrophobic residue. (F) Inner row α-helices of ankyrinR and PUM repeats superimposed in the same coordinate system with amino acid i at the origin, vector C iα 1 C iβ parallel to the X axis, and peptide group (i 1, i) in XZ plane. If positions (i 1, i) of both helices coincide, edges (iþ 1, iþ 2), (iþ 2, iþ 3) and (iþ 3, iþ 4) show different angles with the plane of the peptide group (i 1, i). AnkyrinR repeats (pdb code 1n11), PUM repeats α2/α3 (pdb code 1ib2), ARM repeats of importin α (pdb code 1bk5), and LRR repeats of internalin A (pdb code 2omt) and ribonuclease inhibitor (pdb codes 1dfj and 2bnh) were used.
group plane (i1, i) is negative, whereas the angle of the edge (iþ 1, iþ2) with the same group is positive. However, angle values vary for α-helices of the different assemblies: the angle of the edge (i 3, i 2) with the peptide group plane (i 1, i) is 23.773.31 for PUM repeats and 35.073.71 in ankyrinR. These observations support the data that the geometry of an α-helix and direction of its edges (as sites of attachment of the preceding and following helices) are key factors important for solenoid orientation. 2.3. Pocket geometry and curvature The helix pocket consists of two layers of amino acids which direct their end groups toward intralayer space (Kurochkina, 2008). Interactions of these groups determine layer and pocket geometry. Since amino acids that contribute groups to intralayer space differ in
AnkyrinR and PUM (Fig. 1F), will the direction of the helical edges differ? Inner row α-helices of ankyrinR and PUM repeats when superimposed in the same coordinate system (amino acid i that occupies position a of antiparallel AB interface at the origin, vector C iα 1 C iβ parallel to the X axis with peptide group (i 1, i) in XZ plane) show that if peptide groups (i 1, i) of both helices coincide, angles, that edges (iþ1, iþ2), (iþ2, iþ3), and (iþ3, iþ4) make with peptide group plane, differ (Fig. 1E) and correlate with the curvature of the assembly. For example, average angle between the edge (iþ3, iþ4) and peptide group (i 1, i) in the inner row helices of ankyrin and PUM repeats is 55.874.61 and 48.272.21 respectively. Therefore, orientation of helical edges is important for curvature of the solenoid. To summarize, a relationship exists between intrinsic properties of an α-helix and chirality and curvature of the helical
Please cite this article as: Kurochkina, N.A., Iadarola, M.J., Helical assemblies: Structure determinants. J. Theor. Biol. (2015), http://dx.doi. org/10.1016/j.jtbi.2015.01.012i
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assembly, (2) influences curvature of the assembly, and (3) depends on the amino acid composition of an α-helix.
T145 L144 N143 142
T L141
N
140
I136 I135
P137
L
D134
138
P137
A139 L138
4. Materials and Methods
L141
D134 A133
I132 Q131
N130
Protein structures from the Brookhaven Data Bank (PDB) were used: ankyrinR/PDB designation 1n11/, PUM/1ib2 2yjy/, ARM repeats of importin α/1bk5/, and LRR repeats of internalin A/2omt/ and ribonuclease inhibitor/1dfj 2bnh/, gankyrins/1ixv 1uoh 1wg0 2dzn 3aji 2dwz 2dvw/, Notch/1yyh 2f8y 2qc9 1ot8/, DARPINs/2v48 2xzd/, cyclin dependent kinase inhibitors/1dc2 1a5e 1bi7 1d9e 2a5e 1mx2 1ihb/, designed ankyrins/1mj0/, TRPV1–4, TRPV6/2eta 2nyj 2rfa 2f37 3jxj 4n5q/, and Huntingtin interacting protein/3eu9/. Coordinate system for superposition of proteins and protein fragments and assignment of positions a–g in leucine zipper nomenclature was applied as described in (Kurochkina, 2008). Molecular graphics and matrix transformation operations were performed with JMOL, an open-source Java viewer for chemical structures in 3D (http://www. jmol.org).
5. Discussion
T450 H449 G448 A447 R446
R446 A445
A444
H443 P440
H442 L441
N443
T439 G438
P440 439
A447
V437
T
K436
Fig. 2. Cylindrical projections of the α-helix backbone of (A) AnkyrinR inner row (pdb code 1n11) and (B) internalin A LRR outer row (pdb code 2omt). Circled residues form interfaces with the preceding (open circle) and following (shaded circle) helices of the same row.
assembly. Patterns of contacts of both assembly types, shown on cylindrical projections (Fig. 2) demonstrate how inversion of contacts at the helix–helix interfaces of the inner and outer rows results in change of the direction of the solenoid. Contact sites of the helix with preceding and following helices in the same row go along the direction of the helix backbone and helix axis in the inner rows but opposite to this direction in the outer rows.
Helical assemblies, from one pair of α-helices to multiple repeats, are essential components of large numbers of protein structural motifs including ankyrin, ARM/HEAT, LRR, TPR, leucine zipper, TIM-barrel, Rossman fold, 4-helix bundle, and transmembrane segments. A row of helices has curvature consistent with the pattern of contacts. In ankyrin, ARM/HEAT, and PUM repeats, the inner row of helices carries the same pattern of contacts that involves edges (i 1, i) and (iþ1, i þ2) to interact with preceding and following helices. Repeats of the outer rows of these proteins and LRR repeats carry the pattern of contacts that involves edges (i 1, i) and (i 3, i 2). These two rows cannot be superimposed: edges (i 3, i 2) and (iþ 1, iþ2) that carry contacts involved in the assembly are mirror images of each other. After the outer row is turned approximately 1801 around the Y axis, an antiparallel orientation is achieved relative to the inner row; these rows have the same curvature and bind each other. The inner row is a ligand of the outer row and vice versa. Specificity of binding of these two rows depends on the arrangement of contacts within each row. Orientation of helical edges relative to each other depends on the amino acids within each pocket. Involvement of the same edges but different amino acids at key positions a and d results in the same curvature direction but different curvature parameter: number of the repeats per turn of the solenoid. This is important for protein–protein and protein–ligand specificity of interaction and contributes to the final stability of the complex as well as signaling through binding of ligands such as RNA or ATP. Chirality of assemblies and enantiomer-selective interactions are involved in many processes from growth of viruses and pathological neurodegenerative fibrils to self-assembly of peptides and conjugated polymers (Aggeli et al., 2001; Yang et al., 2013; Marvin et al., 2014; Stephens, 2014). Understanding determinants that govern assembly contributes to our understanding of these and many other phenomena.
3. Conclusion Pocket geometry of an α-helix is a very important characteristic that contributes to chirality and curvature of the helical assembly: orientation of the helical edges (1) determines direction of the
One sentence summary Arrangement of amino acids at helical edges determines handedness of the helical assembly.
Please cite this article as: Kurochkina, N.A., Iadarola, M.J., Helical assemblies: Structure determinants. J. Theor. Biol. (2015), http://dx.doi. org/10.1016/j.jtbi.2015.01.012i
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Chou, K.C., Zhang, C.T., Maggiora, G.M., 1997. Disposition of amphiphilic helices in heteropolar environments. Proteins: Struct. Funct. Genet. 28, 99–108. Crick, F., 1953. Acta Crystallogr. 6, 689. deWit, J., Hong, W., Luo, L., Ghosh, A., 2011. Role of leucine-rich repeat proteins in the development and function of neural circuits. Annu. Rev. Cell Dev. Biol. 27, 697–729. Gaudet, R., 2008. A primer on ankyrin repeat function in TRP channels and beyond. Mol. Biosyst. 4, 372–379. Gerritsen, M., Nemethy, G., Scheraga, H.A., 1985. Energetics of multi-helix interactions in protein folding: application to myoglobin. Biopolymers 24, 1271–1293. Holzer, P., Izzo, A.A., 2014. The pharmacology of TPR channels. Br. J. Pharmacol. 171, 2469–2473. Kohn, W.D., Mant, C.T., Hodges, R.S., 1977. α-Helical protein assembly motifs. J. Biol. Chem. 272, 2583–2586. Kurochkina, N., 2008. Specific sequence combinations at parallel and antiparallel helix–helix interfaces. J. Theor. Biol. 255, 188–198. Kurochkina, N., Choekyi, T., 2011. Helix–helix interfaces and ligand binding. J. Theor. Biol. 283, 92–102. Kurochkina, N., 2010. Helix–helix interactions and their impact on protein motifs and assemblies. J. Theor. Biol. 284, 585–592. Latorre, R., Zaelzer, C., Brauchi, S., 2009. Structure-functional intimacies of transient receptor potential channels. Q. Rev. Biophys. 42, 201–246. Lishko, P.V., Procko, E., Jin, X., Phelps, C.B., Gaudet, R., 2007. The ankyrin repeats of TRPV1 bind multiple ligands and modulate channel sensitivity. Neuron 54, 905–918. Marvin, D.A., Symmons, M.F., Strauss, S.K., 2014. Structure and assembly of filamentous bacteriophages. Prog. Biophys. Mol. Biol. 114, 80–122. Mosavi, L.K., Minor, D.L., Peng, Z., 2002. Consensus-derived structural determinants of the ankyrin repeat motif. Proc. Natl. Acad. Sci. USA 99, 16031–16034. OuYang, B., Xie, S., Berardi, M.J., Zhao, X.M., Dev, J., Yu, W., Sun, B., Chou, J.J., 2013. Unusual architecture of the p7 channel from hepatitis C virus. Nature 498, 521–525. Pauling, L., Corey, R.B., Branson, H.R., 1951. The structure of proteins: two hydrogenbonded helical configurations of the polypeptide chain. Proc. Natl. Acad. Sci. USA 37, 205–211. Sanders, S.S., Mui, K.K.N., Sutton, L.M., Hayden, M.R., 2014. Identification of binding sites in Huntingtin for the Huntingtin interacting proteins HIP14 and HIP14L. Plos One 28, e90669. Sawyer, N., Chen, J., Regan, L., 2013. All repeats are not equal: a module-based approach to guide repeat protein design. J. Mol. Biol. 425, 1826–1838. Scheraga, H.A., Chou, K.C., Nemethy, G., 1982. In: Srinivasan, R., Sarma, R.H. (Eds.), Conformation in Biology. Adenine Press, Gilderland, NY. Schnell, J.R., Chou, J.J., 2008. Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451, 591–595. Schulz, G.E., Schirmer, R.H., 1982. In: Cantor, C.R. (Ed.), Principles of Protein Structure. Stephens, M.J., 2014. Materials science. How shape affects microtubule and nanoparticle assembly. Science 343, 981–982. Stumpp, M.T., Binz, H.K., Amstutz, P., 2008. DARPins: a new generation of protein therapeutics. Drug Discov. Today 13, 695–701. Utreras, E., Prochaskova, M., Terse, A., Gross, J., Keller, J., Iadarola, M.J., Kulkarni, A.B., 2013. TGF-β1 sensitizes TRPV1 through Cdk5 signaling in odontoblast-like cells. Mol. Pain 9, 24–38. Yang, Y., Zhang, Y., Wei, Z., 2013. Supramolecular helices: chirality transfer from conjugated molecules to structures. Adv. Mater. 25, 6039–6049. Zhou, G.P., 2011a. The disposition of the LZCC protein residues in wenxiang diagram provides new insights into the protein–protein interaction mechanism. J. Theor. Biol. 284, 142–148. Zhou, G.P., 2011b. The structural determinations of the leucine zipper coiled-coil domains of the cGMP-dependent protein kinase I alpha and its interaction with the myosin binding subunit of the myosin light chains phosphase. Proteins Pept. Lett. 18, 966–978. Zhou, G.P., Huang, R.B., 2013. The pH-triggered conversion of the PrP(c) to PrP(sc.). Curr. Top. Med. Chem. 13, 1152–1163.
Please cite this article as: Kurochkina, N.A., Iadarola, M.J., Helical assemblies: Structure determinants. J. Theor. Biol. (2015), http://dx.doi. org/10.1016/j.jtbi.2015.01.012i
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Journal of Theoretical Biology journal homepage: www.elsevier.com/locate/yjtbi
Helical assemblies: Structure determinants Natalya A. Kurochkina a,n, Michael J. Iadarola b,1 a
The School of Theoretical Modeling, 1629 K St NW s 300, Washington, DC 20006, United States Anesthesia Section, Department of Periorative Medicine, Clinical Center, NIH, Building 10, Room 2C401, 10 Center Drive, MSC 1510, Bethesda, MD 20892, United States
b
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
Pocket geometry of α-helix contributes to chirality and curvature of the helical assembly. Orientation of helical edges determines direction of the assembly and influences curvature of the assembly. Amino acid composition of α-helix determines its pocket geometry.
art ic l e i nf o
a b s t r a c t
Article history: Received 22 September 2014 Received in revised form 12 January 2015 Accepted 14 January 2015
Protein structural motifs such as helical assemblies and α⧸β barrels combine secondary structure elements with various types of interactions. Helix–helix interfaces of assemblies – Ankyrin, ARM/HEAT, PUM, LRR, and TPR repeats – exhibit unique amino acid composition and patterns of interactions that correlate with curvature of solenoids, surface geometry and mutual orientation of the helical edges. Inner rows of ankyrin, ARM/HEAT, and PUM-HD repeats utilize edges (i 1, i) and (iþ 1, i þ2) for the interaction of the given α-helix with preceding and following helices correspondingly, whereas outer rows of these proteins and LRR repeats invert this pattern and utilize edges (i 1, i) and (i 3, i 2). Arrangement of contacts observed in protein ligands that bind helical assemblies has to mimic the assembly pattern to provide the same curvature as a determinant of binding specificity. These characteristics are important for understanding fold recognition, specificity of protein–protein interactions, and design of new drugs and materials. & 2015 Published by Elsevier Ltd.
Keywords: Protein conformation Repeats Helix–helix interface Assembly Enantiomer-selective ligand binding
1. Introduction Helical assemblies are essential modules of many cellular processes that regulate events of transcription, sensory transduction, development, recognition, and communication (Andrade et al., 2001; deWit et al., 2011; Blatch and Lassle, 1999; Sawyer et al., 2013). As mediators of protein–protein interactions and cell signaling, helical structures provide a basis for our understanding n
Corresponding author. Tel.: þ 1 240 381 2383. E-mail addresses: [email protected] (N.A. Kurochkina), [email protected] (M.J. Iadarola). 1 Tel.: þ 1 301 496 2758.
of many pathologies such as mental, degenerative and immune system diseases, cancer, and inflammation (Utreras et al., 2013; Lishko et al., 2007; Sanders et al., 2014; Holzer and Izzo, 2014; Latorre et al., 2009). Many helix bundles have been shown to be vital for the development of drugs against influenza, obesity/ diabetes, hepatitis C virus and other diseases (Schnell and Chou, 2008; Berardi et al., 2011; OuYang et al., 2013). Designed repeat proteins have the ability to bind their specific targets and provide drug candidates for future treatments (Stumpp et al., 2008; Abil et al., 2014). Structure of the helical assembly can contain one or more rows of stacked helices that form a solenoid, a helix of helices. Each type of the repeat – Ankyrin, ARM/HEAT, Pumilio homology domain (PUM-HD), leucine rich repeat (LRR), or
http://dx.doi.org/10.1016/j.jtbi.2015.01.012 0022-5193/& 2015 Published by Elsevier Ltd.
Please cite this article as: Kurochkina, N.A., Iadarola, M.J., Helical assemblies: Structure determinants. J. Theor. Biol. (2015), http://dx.doi. org/10.1016/j.jtbi.2015.01.012i
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tetratricopeptide (TPR) – exhibits unique properties, characteristic amino acid composition (Andrade et al., 2001; deWit et al., 2011; Blatch and Lassle, 1999; Sawyer et al., 2013), and groups of consensus sequences (Mosavi et al., 2002; Gaudet, 2008). The structure of the α-helix organizes the protein backbone in a specific hydrogen bonding pattern (Pauling et al., 1951). Arrangements and energetics of interactions of α-helices, β-sheets, and loops have been extensively studied with model peptides (Chou et al., 1988, 1989, 1990a, 1990b) and proteins including 8α/8β barrels (Chou and Carlacci, 1991), α-helix bundles (Carlacci, 1990b, 1990c, 1991; Carlacci and Maggiora., 1991; Chou et al., 1992a, 1992b), leucine zippers (Chou, 1992), and globins (Gerritsen et al., 1985). Packing of secondary structure elements “knobs into holes” (Crick, 1953), complementarity of interacting surfaces (Chothia et al., 1981), hydrogen bonding and van der Waals interactions contribute to specificity and stability of protein molecules and energy of hydrophobic and electrostatic interactions (Scheraga et al., 1982; Schulz and Schirmer, 1982; Chou et al., 1983, 1984; Carlacci, 1990a, 1991). Formation of pathogenic β-sheet aggregates from α-helix prion proteins demonstrates polypeptide chain conformational transitions under various conditions (Zhou, 2011b; Zhou and Huang, 2013). The hydrophobic and hydrophilic environment of α-helices is one of the major factors influencing their structural properties (Chou et al., 1997). The distribution of amino acids at the helical surfaces of leucine zipper dimeric molecular complexes clearly shows clustering of hydrophobic residues at interface positions a and d and hydrophilic residues at the interface with solvent (Chou et al., 1990a, 1997, 2011; Zhou, 2011a). As an α-helix binds more ligands, hydrophobic patches expand so that adjacent edges become involved in helix–helix interactions. In membrane helices or helices surrounded by other secondary structure elements, all edges are hydrophobic since they are not exposed to polar solvent. Hydrophobic interactions determined by specific amino acid combinations are important structural determinants of these oligomers. Amino acid combinations characteristic for each type of helix– helix interface and arrangement of α-helices in proteins show good correlation (Kurochkina, 2008; Kurochkina and Choekyi, 2011). Specific combinations at particular helical edges are important for the shape of the assembly as was previously shown for 8α/ 8β TIM-barrel proteins and 4-α-helix subunits of tobacco mosaic virus (Kurochkina, 2010). In the present work, we demonstrate that arrangement of amino acids at the helical edges and specific amino acid combinations of helix–helix interfaces can distinguish one type of helical assembly from another. Inner rows of ankyrin, ARM/HEAT, and PUM-HD repeats utilize edges (i 1, i) and (iþ1, iþ2) for the interaction of the given α-helix with preceding and following helices, respectively, whereas outer rows of these proteins and LRR repeats invert this pattern and utilize edges (i 1, i) and (i 3, i 2). The reason that this inversion of contacts leads to the change in handedness of the assembly can be explained by the geometry of the helical surface and mutual orientation of the helical edges. Each of the two different contact patterns corresponds to a unique helix arrangement. The new approach elaborated can be used to address mechanisms of action of protein molecules, prediction of specific protein–protein interactions, fold recognition, and design of drugs, nanostructures and nanomaterials.
containing peptide groups of hydrogen-bonded residues. A helical edge that contains Cα atoms of the two consecutive amino acids together with all atoms located between α-carbons forms such a plane. An edge can be designated by two consecutive Cα atoms, for example, (i, iþ1) or (iþ1, i þ2) (Fig. 1A). Edges and peptide planes are important for determining both the α-helix shape and recognition of binding surfaces by secondary structure elements. The helix–helix interface is formed by amino acids located mainly at the conserved core positions a and d and less conserved but more exposed positions e and g in leucine zipper nomenclature (Kohn et al., 1977). Each type of the repeats assembly has a repeat unit: a pair of α-helices and a β-hairpin (ankyrin), two (HEAT, TPR) or three (ARM) α-helices, or α-helix and β-strand (LRR) stacked so that they form one or two rows of α-helices (Fig. 1B and C). In the ankyrin repeat molecules, each inner row helix (A) forms an antiparallel interface with outer row helix (B) and two parallel interfaces, one with preceding (A0 ) and one with following (A00 ) helices. As a result, AB, AA0 , and AA00 interfaces are observed in the inner row, and BB0 and BB00 interfaces in the outer row (Fig. 1C). A similar arrangement of helices is present in PUM and ARM/HEAT repeats but they differ in the number of repeats per helical turn, interhelical angles, and structure of the repeat unit. In ARM/HEAT repeats, B helices form an inner (concave) row whereas A helices form an outer (convex) row. In the LRR repeat unit, outer row helices wrap around an inner row β-sheet and a second row of helices (Fig. 1B). Although outer rows of ankyrin, ARM/HEAT, PUM, and LRR repeats have a similar organization (Fig. 1B and C), the direction of the assembly is opposite to that of inner rows. These two types of assemblies cannot be superimposed. The difference in the outline of each assembly can be clearly seen if positions a of the row helices of the two assembly types are shown in the same coordinate system (Fig. 1D). This coordinate system is selected so that the α-carbon of amino acid at position a is at the origin, the peptide group between residue at position a and residue at position g preceding a is in XZ plane, vector from Cα at position g to Cβ at position a is parallel to the X axis, and the negative end of the Y-axis points toward the interacting helix (Kurochkina, 2008). Coordinates of each row of helices are transformed so that position a of the N-terminal helix interface with the following helix is at the origin, and peptide group of the residues at positions g and a is in XZ plane. All consecutive helices of the row will follow in the negative Y direction. We can see that inner and outer rows follow opposite X axis directions. Assignment of positions a, d, e, and g to each helix–helix interface (Fig. 1E) reveals that contacts of the central helix with three surrounding helices follow a particular pattern that is repeated at each ankyrin unit. This pattern differs from the pattern of other helix–helix interfaces. For instance, parallel interfaces of the TIMbarrel proteins utilize edges (i, iþ1) and (iþ5, iþ6) to contact the preceding and following helices (Kurochkina, 2010), whereas the inner rows of ankyrin repeats use (i 1, i) and (iþ 1, iþ 2). This same pattern of contacts is observed at the helix–helix interfaces of the inner rows of ARM/HEAT and PUM-HD assemblies. However, in the outer rows of these proteins and LRR repeats, the pattern of contacts is inverted: edges (i1, i) and (i 3, i 2) are involved in contacts of the central helix with the preceding and following helices. How does this inversion result in the change of the direction of the solenoid producing two types of assemblies that cannot be superimposed?
2. Results
2.2. Pocket geometry and chirality
2.1. Solenoid structures and patterns of interactions
If we draw a plane perpendicular to the peptide group plane of the residues (i 1, i), we can see that the edges (i 3, i 2) and (iþ1, iþ 2) are mirror images of each other (Fig. 1A). Conserved feature of any α-helix is that angle of the edge (i 3, i 2) with the peptide
Each peptide group comprises two amino acids joined by a peptide bond. The surface of an α-helix is shaped by planes
Please cite this article as: Kurochkina, N.A., Iadarola, M.J., Helical assemblies: Structure determinants. J. Theor. Biol. (2015), http://dx.doi. org/10.1016/j.jtbi.2015.01.012i
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Fig. 1. Helical assemblies. (A) Helical wheel. Interactions of the given helix (A) with preceding (A0 0 ) and following (A0 ) helices involve edges (i 1, i; □) and (iþ 1, iþ 2) in the ankyrin inner row ( ), PUM ( ), and ARM/HEAT ( ) repeats but (i 1, i; □) and (i 3, i 2) in LRR repeats ( ) and ankyrin outer row ( ). Leading edges are designated by filled boxes. Edges (i 3, i 2) and (i þ1, iþ 2) are nonsuperimposable mirror images of each other. (B) LRR repeats of Internalin A (INLA). (C) Ankyrin repeats of ankyrinR inner row. (D) The line connecting positions a of the repeats in the XY plane. The lowest and highest points in the Z direction are shown by empty and filled enlarged markers correspondingly. (E) Comparison of characteristic pattern of contacts in ankyrinR repeats and internalin A LRR repeats. Amino acid sequence alignment of the repeats and their consensus sequences; α-helices are boxed; ψ–hydrophobic residue. (F) Inner row α-helices of ankyrinR and PUM repeats superimposed in the same coordinate system with amino acid i at the origin, vector C iα 1 C iβ parallel to the X axis, and peptide group (i 1, i) in XZ plane. If positions (i 1, i) of both helices coincide, edges (iþ 1, iþ 2), (iþ 2, iþ 3) and (iþ 3, iþ 4) show different angles with the plane of the peptide group (i 1, i). AnkyrinR repeats (pdb code 1n11), PUM repeats α2/α3 (pdb code 1ib2), ARM repeats of importin α (pdb code 1bk5), and LRR repeats of internalin A (pdb code 2omt) and ribonuclease inhibitor (pdb codes 1dfj and 2bnh) were used.
group plane (i1, i) is negative, whereas the angle of the edge (iþ 1, iþ2) with the same group is positive. However, angle values vary for α-helices of the different assemblies: the angle of the edge (i 3, i 2) with the peptide group plane (i 1, i) is 23.773.31 for PUM repeats and 35.073.71 in ankyrinR. These observations support the data that the geometry of an α-helix and direction of its edges (as sites of attachment of the preceding and following helices) are key factors important for solenoid orientation. 2.3. Pocket geometry and curvature The helix pocket consists of two layers of amino acids which direct their end groups toward intralayer space (Kurochkina, 2008). Interactions of these groups determine layer and pocket geometry. Since amino acids that contribute groups to intralayer space differ in
AnkyrinR and PUM (Fig. 1F), will the direction of the helical edges differ? Inner row α-helices of ankyrinR and PUM repeats when superimposed in the same coordinate system (amino acid i that occupies position a of antiparallel AB interface at the origin, vector C iα 1 C iβ parallel to the X axis with peptide group (i 1, i) in XZ plane) show that if peptide groups (i 1, i) of both helices coincide, angles, that edges (iþ1, iþ2), (iþ2, iþ3), and (iþ3, iþ4) make with peptide group plane, differ (Fig. 1E) and correlate with the curvature of the assembly. For example, average angle between the edge (iþ3, iþ4) and peptide group (i 1, i) in the inner row helices of ankyrin and PUM repeats is 55.874.61 and 48.272.21 respectively. Therefore, orientation of helical edges is important for curvature of the solenoid. To summarize, a relationship exists between intrinsic properties of an α-helix and chirality and curvature of the helical
Please cite this article as: Kurochkina, N.A., Iadarola, M.J., Helical assemblies: Structure determinants. J. Theor. Biol. (2015), http://dx.doi. org/10.1016/j.jtbi.2015.01.012i
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assembly, (2) influences curvature of the assembly, and (3) depends on the amino acid composition of an α-helix.
T145 L144 N143 142
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4. Materials and Methods
L141
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Protein structures from the Brookhaven Data Bank (PDB) were used: ankyrinR/PDB designation 1n11/, PUM/1ib2 2yjy/, ARM repeats of importin α/1bk5/, and LRR repeats of internalin A/2omt/ and ribonuclease inhibitor/1dfj 2bnh/, gankyrins/1ixv 1uoh 1wg0 2dzn 3aji 2dwz 2dvw/, Notch/1yyh 2f8y 2qc9 1ot8/, DARPINs/2v48 2xzd/, cyclin dependent kinase inhibitors/1dc2 1a5e 1bi7 1d9e 2a5e 1mx2 1ihb/, designed ankyrins/1mj0/, TRPV1–4, TRPV6/2eta 2nyj 2rfa 2f37 3jxj 4n5q/, and Huntingtin interacting protein/3eu9/. Coordinate system for superposition of proteins and protein fragments and assignment of positions a–g in leucine zipper nomenclature was applied as described in (Kurochkina, 2008). Molecular graphics and matrix transformation operations were performed with JMOL, an open-source Java viewer for chemical structures in 3D (http://www. jmol.org).
5. Discussion
T450 H449 G448 A447 R446
R446 A445
A444
H443 P440
H442 L441
N443
T439 G438
P440 439
A447
V437
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K436
Fig. 2. Cylindrical projections of the α-helix backbone of (A) AnkyrinR inner row (pdb code 1n11) and (B) internalin A LRR outer row (pdb code 2omt). Circled residues form interfaces with the preceding (open circle) and following (shaded circle) helices of the same row.
assembly. Patterns of contacts of both assembly types, shown on cylindrical projections (Fig. 2) demonstrate how inversion of contacts at the helix–helix interfaces of the inner and outer rows results in change of the direction of the solenoid. Contact sites of the helix with preceding and following helices in the same row go along the direction of the helix backbone and helix axis in the inner rows but opposite to this direction in the outer rows.
Helical assemblies, from one pair of α-helices to multiple repeats, are essential components of large numbers of protein structural motifs including ankyrin, ARM/HEAT, LRR, TPR, leucine zipper, TIM-barrel, Rossman fold, 4-helix bundle, and transmembrane segments. A row of helices has curvature consistent with the pattern of contacts. In ankyrin, ARM/HEAT, and PUM repeats, the inner row of helices carries the same pattern of contacts that involves edges (i 1, i) and (iþ1, i þ2) to interact with preceding and following helices. Repeats of the outer rows of these proteins and LRR repeats carry the pattern of contacts that involves edges (i 1, i) and (i 3, i 2). These two rows cannot be superimposed: edges (i 3, i 2) and (iþ 1, iþ2) that carry contacts involved in the assembly are mirror images of each other. After the outer row is turned approximately 1801 around the Y axis, an antiparallel orientation is achieved relative to the inner row; these rows have the same curvature and bind each other. The inner row is a ligand of the outer row and vice versa. Specificity of binding of these two rows depends on the arrangement of contacts within each row. Orientation of helical edges relative to each other depends on the amino acids within each pocket. Involvement of the same edges but different amino acids at key positions a and d results in the same curvature direction but different curvature parameter: number of the repeats per turn of the solenoid. This is important for protein–protein and protein–ligand specificity of interaction and contributes to the final stability of the complex as well as signaling through binding of ligands such as RNA or ATP. Chirality of assemblies and enantiomer-selective interactions are involved in many processes from growth of viruses and pathological neurodegenerative fibrils to self-assembly of peptides and conjugated polymers (Aggeli et al., 2001; Yang et al., 2013; Marvin et al., 2014; Stephens, 2014). Understanding determinants that govern assembly contributes to our understanding of these and many other phenomena.
3. Conclusion Pocket geometry of an α-helix is a very important characteristic that contributes to chirality and curvature of the helical assembly: orientation of the helical edges (1) determines direction of the
One sentence summary Arrangement of amino acids at helical edges determines handedness of the helical assembly.
Please cite this article as: Kurochkina, N.A., Iadarola, M.J., Helical assemblies: Structure determinants. J. Theor. Biol. (2015), http://dx.doi. org/10.1016/j.jtbi.2015.01.012i
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