Effects of side-chain characteristics on stability and oligomerization state of a de Novo -designed model coiled-coil: 20 amino acid substitutions in position “d” 1 1Edited by P. E. Wright

Tripet, Brian; Wagschal, Kurt; Lavigne, Pierre; Mant, Colin T.; Hodges, Robert S.

doi:10.1006/jmbi.2000.3866

Cited by 240 publications

(294 citation statements)

References 64 publications

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“…The primary stabilizing interactions are the hydrophobic interactions in the core (34,37), electrostatic attractions across the interface (5,7,9,16), and helical propensity (16,(38)(39)(40)(41). Valine and leucine were chosen for positions a and d, as they are known to form a highly stable hydrophobic core (35,36,42,43). We have used electrostatic interactions at positions e and g to control specificity for heterodimer versus homodimer formation, an approach successfully exploited in both natural and de novo sequences (10-13, 16-17, 22) One strand of the coiled-coil, the E coil, has glutamic acid (which is preferable to aspartic acid because of its higher helical propensity) at all e and g positions, while the complementary K coil has lysine at all e and g positions ( Figure 1).…”

Section: Resultsmentioning

confidence: 99%

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

et al. 2003

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We have de novo designed a heterodimeric coiled-coil formed by two peptides as a capture/ delivery system that can be used in applications such as affinity tag purification, immobilization in biosensors, etc. The two strands are designated as K coil (KVSALKE heptad sequence) and E coil (EVSALEK heptad sequence), where positively charged or negatively charged residues occupy positions e and g of the heptad repeat. In this study, for each E coil or K coil, three peptides were synthesized with lengths varying from three to five heptads. The effect of the chain length of each partner upon the kinetic and thermodynamic constants of interaction were determined using a surface plasmon resonance-based biosensor. Global fitting of the interactions revealed that the E5 coil interacted with the K5 coil according to a simple binding model. All the other interactions involving shorter coils were better described by a more complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. The affinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to 30 µM (E3/K3). From these K d values, we were able to determine the free energy contribution of each heptad, depending on its relative position within the coiled-coils. We found that the free energy contribution of a heptad occupying a central position was 3-fold higher than that of a heptad at either end of the coiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerable flexibility for protein engineering and biotechnological applications.The R-helical coiled-coil is an oligomerization domain that is naturally present in a wide variety of proteins such as transcription factors, cytoskeletal proteins, motor proteins, and viral fusion proteins (1-3). The structural properties of coiled-coils have been extensively characterized from numerous high-resolution crystal and NMR structures (3-5). The formation of coiled-coils requires the wrapping of two or more amphipathic R-helices around each other in a lefthanded supercoil fashion; the helices may be aligned in either a parallel or antiparallel manner. The R-helices composing the coiled-coil structure are defined by a heptad repeat (denoted abcdefg) in which hydrophobic residues occupy the positions a and d and pack in a characteristic "knobs-intoholes" manner (6), forming the hydrophobic core ( Figure 1). Many studies (1,(7)(8)(9)(10)(11)(12) emphasized the role of charged residues (typically in the e and g positions) in controlling the specificity of oligomerization and opened up the way to the de novo design of heterodimeric coiled-coils (13-17). Their relatively small size, in addition to their well-defined structure, make coiled-coils a very attractive system for protein engineering and biotechnological applications (5, 18) such as the construction of miniaturized antibodies, the control of signaling protein domain oligomerization, and the specific targeting of GFP to cytoskeletal proteins (see ref 4 for ...

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Section: Resultsmentioning

confidence: 99%

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

et al. 2003

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“…These domains are predicted to have a high probability (Ͼ98%) of forming coiled-coil structures as determined by the COILS algorithm (30). Each exhibits the characteristic heptad repeat motif (31) with hydrophobic residues at positions a and d and charged or polar residues at e and g. Residues a and d provide the interface between interacting ␣-helices, and different modes of interaction are possible. An additional putative coiled-coil domain (probability Ͼ95%) is found in WbdD O9a only (Fig.…”

Section: Conserved Motifs In the Wbdd Proteins Implicate Them In The mentioning

confidence: 99%

Nonreducing Terminal Modifications Determine the Chain Length of Polymannose O Antigens of Escherichia coli and Couple Chain Termination to Polymer Export via an ATP-binding Cassette Transporter

Clarke

Cuthbertson²,

Whitfield

2004

Journal of Biological Chemistry

105

145

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The chain length of bacterial lipopolysaccharide O antigens is regulated to give a modal distribution that is critical for pathogenesis. This paper describes the process of chain length determination in the ATP-binding cassette (ABC) transporter-dependent pathway, a pathway that is widespread among Gram-negative bacteria. Escherichia coli O8 and O9/O9a polymannans are synthesized in the cytoplasm, and an ABC transporter exports the nascent polymer across the inner membrane prior to completion of the LPS molecule. The polymannan O antigens have nonreducing terminal methyl groups. The 3-O-methyl group in serotype O8 is transferred from S-adenosylmethionine by the WbdD O8 enzyme, and this modification terminates polymerization. Methyl groups are added to the O9a polymannan in a reaction dependent on preceding phosphorylation. The bifunctional WbdD O9a catalyzes both reactions, but only the kinase activity controls chain length. Chain termination occurs in a mutant lacking the ABC transporter, indicating that it precedes export. An E. coli wbdD O9a mutant accumulated O9a polymannan in the cytoplasm, indicating that WbdD activity coordinates polymannan chain termination with export across the inner membrane.

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“…1, a and b) with positions a and d in each heptad repeat occupied by hydrophobic residues (20,31). Leucine provides the most thermodynamically stable residue at position d (32). However, significant deviations in amino acid sequence from this "classical" leucine zipper motif may exist, for example, deletions or insertions of individual residues within the heptad repeat or the presence of polar residues at position a or d (22,31,32,34).…”

Section: Resultsmentioning

confidence: 99%

“…1, a and b, between residues 513 and 548 of the murine BK channel variant mbr5 (accession number GI:347144 (9)) contains five heptad repeats downstream of residues that contribute to the proposed "fixed interface" of the BK channel regulator of K channel conductance (RCK, or tetramerization) domain (26 -28). Although the second d residue of the five-heptad LZ1 repeat is glutamine (Q) and the fifth a residue is non-hydrophobic, the stability of "prototypical" LZ domains with a Gln (Q) residue at a single d position is, paradoxically, not significantly compromised compared with isoleucine or valine substitutions (32). Comparison of the amino acid sequence of mammalian BK channels with the structure of the RCK domain in calciumactivated potassium (MthK) channels from Methanobacterium thermoautotrophicum suggests that LZ1 forms the ␣G helix and also contributes to an extended linker region between the ␣G helix and ␤G strand (26 -28).…”

Section: Resultsmentioning

confidence: 99%

Leucine Zipper Domain Targets cAMP-dependent Protein Kinase to Mammalian BK Channels

Tian

Coghill

MacDonald

et al. 2003

Journal of Biological Chemistry

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Large conductance, calcium-and voltage-activated potassium (BK) channels control excitability in many tissues and are regulated by several protein kinases and phosphatases that remain associated with the channels in cell-free patches of membrane. Here, we report the identification of a highly conserved, non-canonical, leucine zipper (LZ1) in the C terminus of mammalian BK channels that is required for cAMP-dependent protein kinase ( Reversible protein phosphorylation represents a fundamental cellular regulatory mechanism to control the activity and function of plasma membrane ion channels (1). The co-ordination, specificity, and compartmentalization of ion channel regulation by reversible protein phosphorylation is facilitated by assembly with signaling complexes comprising cognate protein kinases and protein phosphatases. Assembly of ion channels with signaling complexes typically results from multiple protein-protein interactions mediated by distinct interaction domains (2) allowing signaling molecules to interact directly with an ion channel or indirectly as part of a higher order complex.Large conductance calcium-and voltage-activated potassium (BK) channels have been widely exploited as models of ion channel regulation by reversible protein phosphorylation; however, the molecular basis for kinase and phosphatase assembly with the BK channel is largely unknown

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Effects of side-chain characteristics on stability and oligomerization state of a de Novo -designed model coiled-coil: 20 amino acid substitutions in position “d” 1 1Edited by P. E. Wright

Cited by 240 publications

References 64 publications

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

Nonreducing Terminal Modifications Determine the Chain Length of Polymannose O Antigens of Escherichia coli and Couple Chain Termination to Polymer Export via an ATP-binding Cassette Transporter

Leucine Zipper Domain Targets cAMP-dependent Protein Kinase to Mammalian BK Channels

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