Dynamic charge interactions create surprising rigidity in the ER/K α-helical protein motif

Sivaramakrishnan, Sivaraj; Spink, Benjamin J.; Sim, Adelene Y. L.; Doniach, Sebastian; Spudich, James A.

doi:10.1073/pnas.0806256105

Cited by 101 publications

(154 citation statements)

References 29 publications

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“…1). Their logic was that a construct containing 835-1,035 did not dimerize and that the region of myosin VI that was originally thought to form a coiled coil has been proposed to be a stable single ␣-helix (SAH) (18,23,24). Thus, Spink et al (18) postulated that the SAH domain forms an extension of the short myosin VI lever arm, enabling the dimer to take large steps on actin.…”

Section: Discussionmentioning

confidence: 99%

Cargo binding induces dimerization of myosin VI

Phichith

Travaglia²,

Yang³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

105

104

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Although myosin VI has properties that would allow it to function optimally as a dimer, full-length myosin VI exists as a monomer in isolation. Based on the ability of myosin VI monomers to dimerize when held in close proximity, we postulated that cargo binding normally regulates dimerization of myosin VI. We tested this hypothesis by expressing a known dimeric cargo adaptor protein of myosin VI, optineurin, and the myosin VI-binding segment from a monomeric cargo adaptor protein, Dab2. In the presence of these adaptor proteins, full-length myosin VI has ATPase properties of a dimer, appears as a dimer in electron micrographs, and moves processively on actin filaments. The results support a model in which cargo binding exposes internal dimerization sequences within full-length myosin VI. Because, unexpectedly, a monomeric fragment of Dab2 triggers dimerization, it would appear that myosin VI is designed to function as a dimer in cells.Dab2 ͉ directionality ͉ motility ͉ optineurin ͉ unconventional myosin W ithin the myosin superfamily there are at least 35 classes of molecular motors that move along actin filaments (1). Myosin VI is the only class of myosin known to move toward the minus-end of actin filaments (2, 3). Myosin VI dimers take large and variable steps on actin (average of 30-36 nm) using a short lever arm and a unique lever arm extension (4-6). Not only is the myosin VI dimer capable of processive movement (i.e., can move as a single molecule) along an actin filament (4-6), it also functions as a load-dependent actin anchoring protein (7). Thus myosin VI can potentially fulfill a number of specialized cell biological functions (8-10). Paradoxically, although these functional features suggest that myosin VI is designed to work in cells as a dimer, myosin VI as isolated from cells is a monomer, and expressed full-length myosin VI is also monomeric (4,5,11).A number of cargo adaptor proteins that recruit myosin VI have been identified (8). For example, it has been demonstrated that optineurin is essential for myosin VI localization to the Golgi complex (12), and binds to a site within the globular tail of myosin VI. Dab2 (13,14) and Sap97 (15) mediate the recruitment of myosin VI to clathrin-coated pits and vesicles, whereas GipC serves this role on uncoated vesicles (16,17).The myosin VI molecule has discrete structural domains, as diagrammed in Fig. 1, using the terminology of Spink et al. (18). We have demonstrated that internal dimerization (probably coiled coil) occurs between residues 913 and 936 (6). However, we noted that this dimerization is weak and forms only if the molecules are held in close proximity (5, 6). We postulated that in vivo dimerization is initiated upon binding of myosin VI to a dimeric cargo, which would then trigger the weak internal dimerization (5, 6).Subsequently, it was shown that a headless myosin VI construct containing the entire tail and cargo-binding domains dimerized with relatively high affinity (18). Thus there may be two separate regions of the myosin VI molecule...

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Section: Discussionmentioning

confidence: 99%

Cargo binding induces dimerization of myosin VI

Phichith

Travaglia²,

Yang³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

105

104

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“…The presence of the guanidinium group may enable Arg to interact simultaneously with the negative Glu residues in both directions. 13 …”

Section: E-r Peptides Have Higher Helix Content Than E-k Peptidesmentioning

confidence: 99%

“…This motif of four Glu residues followed by a combination of four Lys and/or Arg residues has been found in a variety of proteins, including caldesmon, 11 myosin X, and myosin VI. 12,13 It has been shown to form long (up to 30 nm), single, stable, and relatively rigid helices (persistence length ¼ 15 nm) in various proteins. [14][15][16] Experimental measurement of helicity using circular dichroism of peptides has been used in conjunction with statistical mechanics models of helixcoil transition 17,18 to develop prediction programs for the helicity of peptides.…”

Section: Introductionmentioning

confidence: 99%

Helicity of short E‐R/K peptides

et al. 2010

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Understanding the secondary structure of peptides is important in protein folding, enzyme function, and peptide-based drug design. Previous studies of synthetic Ala-based peptides (>12 a.a.) have demonstrated the role for charged side chain interactions involving Glu/Lys or Glu/Arg spaced three (i, i 1 3) or four (i, i 1 4) residues apart. The secondary structure of short peptides (<9 a.a.), however, has not been investigated. In this study, the effect of repetitive Glu/Lys or Glu/Arg side chain interactions, giving rise to E-R/K helices, on the helicity of short peptides was examined using circular dichroism. Short E-R/K-based peptides show significant helix content. Peptides containing one or more E-R interactions display greater helicity than those with similar E-K interactions. Significant helicity is achieved in Arg-based E-R/K peptides eight, six, and five amino acids long. In these short peptides, each additional i 1 3 and i 1 4 salt bridge has substantial contribution to fractional helix content. The E-R/K peptides exhibit a strongly linear melt curve indicative of noncooperative folding. The significant helicity of these short peptides with predictable dependence on number, position, and type of side chain interactions makes them an important consideration in peptide design.

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“…Sivaramakrishnan et al (18) performed a replica exchange MD simulation on a (E 4 K 4 ) 2 peptide (Fig. 1c).…”

Section: Stability Of Er/k ␣-Helicesmentioning

confidence: 99%

Harnessing the Unique Structural Properties of Isolated α-Helices

Swanson

Sivaramakrishnan

2014

Journal of Biological Chemistry

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The ␣-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 ␣-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 ␣-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 ϳ4 consecutive glutamic acids followed by ϳ4 consecutive basic arginine or lysine (R/K) residues. The ER/K ␣-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. Coiled-coil or Single ␣-Helical (SAH) Domain?Until recently, SAH 2 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). Among the multiple folds in globular proteins that stabilize ␣-helices, the coiled-coil motif has been extensively characterized and in general is the most predictable form of tertiary protein structure (2). In the coiled-coil motif, two or more ␣-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 ␣-helix in a coiled-coil is divided into heptads (7 residues) that form nearly two complete ␣-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 ␣-helix, 60°a part 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 ␣-helix compatible with a tight intermolecular interaction between polypeptides with the same or similar heptad pattern (2). However, often polar or charged residues occupy the a and d positions, leading to local destabilization of the coiled-coil...

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Dynamic charge interactions create surprising rigidity in the ER/K α-helical protein motif

Cited by 101 publications

References 29 publications

Cargo binding induces dimerization of myosin VI

Cargo binding induces dimerization of myosin VI

Helicity of short E‐R/K peptides

Harnessing the Unique Structural Properties of Isolated α-Helices

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