Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides^,

Abstract

Cavities and clefts are frequently important sites of interaction between natural enzymes or receptors with their corresponding substrate or ligand molecules and exemplify the types of molecular surfaces that would facilitate engineering artificial catalysts and receptors. Even so, structural characterizations of designed cavities are rare. To address this issue, we performed a systematic study of the structural effects of single amino acid substitutions within the hydrophobic cores of tetrameric coiled-coil peptides. Peptides containing single glycine, serine, alanine, or threonine amino acid substitutions at the buried L9, L16, L23, and I26 hydrophobic core positions of a GCN4-based sequence were synthesized and studied by solution-phase and crystallographic techniques. All peptides adopt the expected tetrameric state and contain tunnels or internal cavities ranging in size from 80 Å 3 to 370 Å 3 . Two closely-related sequences containing an L16G substitution, one of which adopts an antiparallel configuration and one of which adopts a parallel configuration, illustrate that cavities of different volumes and shapes can be engineered from identical core substitutions. Finally, we demonstrate that two of the peptides (L9G and L9A) bind the small molecule iodobenzene when present during crystallization, leaving the general peptide quaternary structure intact but altering the local peptide conformation and certain superhelical parameters. These high-resolution descriptions of varied molecular surfaces within solvent-occluded internal cavities illustrate the breadth of design space available in even closely-related peptides and offer valuable models for the engineering of de novo helical proteins.A significant challenge frustrating the de novo design of functional molecules is that of precisely constructing molecular surfaces able to suitably participate in desired molecular recognition events. Internal cavities, relatively buried clefts, and tunnels are frequently important sites of interaction between natural enzymes or receptors with their corresponding substrate or ligand molecules, and the ability to engineer such surfaces would facilitate the successful design of highly-selective artificial catalysts and receptors. Mutagenesis of natural proteins to create or alter internal cavities has been used for such purposes as redesigning † We thank the National Institutes of Health for financial support (GM52190). JER, JMAG, and LJL thank the Wellcome Trust (061454/ Z/00/Z), the Spanish MCYT, and NSF, respectively, for fellowships. § Coordinates have been deposited in the RCSB Protein Data Bank: GCN4-pLI, 1UO2; L9G, 1UO3; L9S, 1UNU; L9A, 1UNT; L9T, 1UNV; L9G+IB, 1UO4; L9A+IB, 1UO5; L23G, 1UNW; L23S 1UNX; I26G, 1UNY; I26S, 1UNZ; I26A, 1UN0; I26T, 1UN1; L16G E20C, 1W5I; L16G E20C Y17H, 2BNI.* Address correspondence to this author. (858) 784-2700 (phone); (858) 784-2798 (fax); ghadiri@scripps.edu (e-mail).. 1 Abbreviations: CD, circular dichroism; SEC, size exclusion chromatography; ABA, acetamidobenzoic acid; MW,...

Coiled Coils at the Edge of Configurational Heterogeneity. Structural Analyses of Parallel and Antiparallel Homotetrameric Coiled Coils Reveal Configurational Sensitivity to a Single Solvent-Exposed Amino Acid Substitution,

“…b The crystallization and structure determination of this peptide has been reported previously (17). b The full-length sequence (residues 1-33), rather than a truncated sequence (residues 2-32), was used for simulation.…”

“…c This variant of GCN4-pLI, containing an amino acid substitution in the hydrophobic core, has previously been shown to crystallize in the parallel configuration (17). Table 3 Decomposition of total potential energies (in kcal/mol) from lowest temperature window (280 K) of lowestaverage-energy simulations of GCN4-pLI (1) and E20C (3).…”

Hydrophobic Interactions Are Key To Drive the Association of Tapasin with Peptide Transporter Subunit TAP2

“…5C) using the cyclic hydrocarbon benzene. Benzene binds efficiently to cellular membranes (45), interacts with hydrophobic protein patches and/or cores, and modulates the arrangement of hydrophobic protein interfaces (19,20). Benzene does not affect the cholesterol-binding properties of digitonin (23), but it specifically targets leucine zippers containing central small residues (19,20) (see Fig.…”

“…In contrast, three highly conserved arginine and aspartate residues located within or directly adjacent to these TMs are not required. Moreover, we demonstrate that the cyclic hydrocarbon benzene known to interfere with the structure and formation of leucine zippers (19,20) specifically disrupts the physical interaction between TAP and TPN even under stabilizing detergent conditions. Based on our data, we conclude that TM1 and TM2 of TAP2 are essential for the functional interaction with TPN/MHC I.…”

Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

“…Large hydrophobic cavities are known from various studies to destabilize the folded state. 32 The energetic penalty for introducing a cavity into a 4 tbA 6 can be estimated using the generally accepted value of 24 cal mol À1 Å À3 for the hydrophobic effect in proteins. 33 The calculated reduction in stability is $4.8 kcal mol À1 , which is in reasonable agreement with the observed decrease in stability compared to fluorinated proteins with similar hydrophobic surface areas.…”