Nine hydrophobic side chains are key determinants of the thermodynamic stability and oligomerization status of tumour suppressor p53 tetramerization domain.

The contribution of almost each amino acid side chain to the thermodynamic stability of the tetramerization domain (residues 326-353) of human p53 has been quantitated using 25 mutants with single-residue truncations to alanine (or glycine). Truncation of either Leu344 or Leu348 buried at the tetramer interface, but not of any other residue, led to the formation of dimers of moderate stability (8-9 kcal/mol of dimer) instead of tetramers. One-third of the substitutions were moderately destabilizing (<3.9 kcal/mol of tetramer). Truncations of Arg333, Asn345 or Glu349 involved in intermonomer hydrogen bonds, Ala347 at the tetramer interface or Thr329 were more destabilizing (4.1-5.7 kcal/mol). Strongly destabilizing (8.8- 11.7 kcal/mol) substitutions included those of Met340 at the tetramer interface and Phe328, Arg337 and Phe338 involved peripherally in the hydrophobic core. Truncation of any of the three residues involved centrally in the hydrophobic core of each primary dimer either prevented folding (Ile332) or allowed folding only at high protein concentration or low temperature (Leu330 and Phe341). Nine hydrophobic residues per monomer constitute critical determinants for the stability and oligomerization status of this p53 domain.     

The dihedral symmetry of the p53 tetramerization domain mandates a conformational switch upon DNA binding.

The p53 tumor suppressor forms stable tetramers, whose DNA binding activity is allosterically regulated. The tetramerization domain is contained within the C-terminus (residues 323-355) and its three-dimensional structure exhibits dihedral symmetry, such that a p53 tetramer can be considered a dimer of dimers. Under conditions where monomeric p53 fails to bind DNA, we studied the effects of p53 C-terminal mutations on DNA binding. Residues 322-355 were sufficient to drive DNA binding of p53 as a tetramer. Within this region residues predicted by the three-dimensional structure to stabilize tetramerization, such as Arg337 and Phe341, were critical for DNA binding. Furthermore, substitution of Leu344 caused p53 to dissociate into DNA binding-competent dimers, consistent with the location of this residue at the dimer-dimer interface. The p53 DNA site contains two inverted repeats juxtaposed to a second pair of inverted repeats. Thus, the four repeats exhibit cyclic-translation symmetry and cannot be recognized simultaneously by four dihedrally symmetric p53 DNA binding domains. The discrepancy may be resolved by flexible linkers between the p53 DNA binding and tetramerization domains. When these linkers were deleted p53 exhibited novel DNA binding properties consistent with an inability to recognize four contiguous DNA repeats. Allosteric regulation of p53 DNA binding may involve repositioning the DNA binding domains from a dihedrally symmetric state to a DNA-bound asymmetric state.     

Molecular determinants of tetramerization in the KcsA cytoplasmic domain

The cytoplasmic C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four-fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD-derived 34-residue peptide associates into a tetramer in a pH-dependent manner (Kamnesky et al., JMB 2012;418:237-247). Here we further investigate the molecular determinants of tetramer formation in the CTD by characterizing the kinetics of monomer-tetramer equilibrium for 10 alanine mutants using NMR, sedimentation equilibrium (SE) and molecular dynamics simulation. NMR and SE concur in finding single-residue contributions to tetramer stability to be in the 0.5 to 3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than electrostatic interactions between residues R147, D149 and E152. In particular, alanine replacement of residue R147, a key contributor to inter-subunit salt bridges, resulted in only a minor effect on tetramer dissociation. Mutations outside of the inter-subunit interface also influenced tetramer stability by affecting the tetramerization on-rate, possibly by changing the inherent helical propensity of the peptide. These findings are interpreted in the context of established paradigms of protein-protein interactions and protein folding, and lay the groundwork for further studies of the CTD in full-length KcsA channels.     

Folding stability modulation of the villin headpiece helical subdomain by 4‐fluorophenylalanine and 4‐methylphenylalanine

HP36, the helical subdomain of villin headpiece, contains a hydrophobic core composed of three phenylalanine residues (Phe47, Phe51, and Phe58). Hydrophobic effects and electrostatic interactions were shown to be the critical factors in stabilizing this core and the global structure. To assess the interactions among Phe47, Phe51, and Phe58 residues and investigate how they affect the folding stability, we implanted 4‐fluorophenylalanine (Z) and 4‐methylphenylalanine (X) into the hydrophobic core of HP36. We chemically synthesized HP36 and its seven variants including four single mutants whose Phe51 or Phe58 was replaced with Z or X, and three double mutants whose Phe51 and Phe58 were both substituted. Circular dichroism and nuclear magnetic resonance measurements show that the variants exhibit a native HP36 like fold, of which F51Z and three double mutants are more stable than the wild type. Molecular modeling provided detailed interaction energy within the phenylalanine residues, revealing that electrostatic interactions dominate the stability modulation upon the introduction of 4‐fluorophenylalanine and 4‐methylphenylalanine. Our results show that these two non‐natural amino acids can successfully tune the interactions in a relatively compact hydrophobic core and the folding stability without inducing dramatic steric effects. Such an approach may be applied to other folded motifs or proteins. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 627–637, 2015.     

Thermal unfolding simulations of a multimeric protein--transition state and unfolding pathways

The folding of an oligomeric protein poses an extra challenge to the folding problem because the protein not only has to fold correctly; it has to avoid nonproductive aggregation. We have carried out over 100 molecular dynamics simulations using an implicit solvation model at different temperatures to study the unfolding of one of the smallest known tetramers, p53 tetramerization domain (p53tet). We found that unfolding started with disruption of the native tetrameric hydrophobic core. The transition state for the tetramer to dimer transition was characterized as a diverse ensemble of different structures using Phi value analysis in quantitative agreement with experimental data. Despite the diversity, the ensemble was still native-like with common features such as partially exposed tetramer hydrophobic core and shifts in the dimer-dimer arrangements. After passing the transition state, the secondary and tertiary structures continued to unfold until the primary dimers broke free. The free dimer had little secondary structure left and the final free monomers were random-coil like. Both the transition states and the unfolding pathways from these trajectories were very diverse, in agreement with the new view of protein folding. The multiple simulations showed that the folding of p53tet is a mixture of the framework and nucleation-condensation mechanisms and the folding is coupled to the complex formation. We have also calculated the entropy and effective energy for the different states along the unfolding pathway and found that the tetramerization is stabilized by hydrophobic interactions.     

Conformational detection of p53’s oligomeric state by FlAsH Fluorescence

The p53 tumor suppressor protein is a critical checkpoint in prevention of tumor formation, and the function of p53 is dependent on proper formation of the active tetramer. In vitro studies have shown that p53 binds DNA most efficiently as a tetramer, though inactive p53 is predicted to be monomeric in vivo. We demonstrate that FlAsH binding can be used to distinguish between oligomeric states of p53, providing a potential tool to explore p53 oligomerization in vivo. The FlAsH tetra-cysteine binding motif has been incorporated along the dimer and tetramer interfaces in the p53 tetramerization domain to create reporters for the dimeric and tetrameric states of p53, though the geometry of the four cysteines is critical for efficient FlAsH binding. Furthermore, we demonstrate that FlAsH binding can be used to monitor tetramer formation in real-time. These results demonstrate the potential for using FlAsH fluorescence to monitor protein-protein interactions in vivo.     

Folding of Tetrameric p53: Oligomerization and Tumorigenic Mutations Induce Misfolding and Loss of Function

The physiologically active form of p53 consists of a tetramer of four identical 393-amino-acid subunits associated via their tetramerization domains (TDs; residues 325-355). One in two human tumors contains a point mutation in the DNA binding domain (DBD) of p53 (residues 94-312). Most existing studies on the effects of these mutations on p53 structure and function have been carried out on the isolated DBD fragment, which is monomeric. Recent structural evidence, however, suggests that DBDs may interact with each other in full-length tetrameric forms of p53. Here, we investigate the effects of tumorigenic DBD mutations on the folding of p53 in its tetrameric form. We employ the construct consisting of DBD and TD (amino acids 94-360). We characterize the stability and conformational state of the tumorigenic DBD mutants R248Q, R249S, and R282Q using equilibrium denaturation and functional assays. Destabilizing mutations cause DBD to misfold when it is part of the p53 tetramer, but not when it is monomeric. This conformation is populated under moderately destabilizing conditions (10 °C in 2 M urea, and at physiological temperature in the absence of denaturant). Under those same conditions, it is not present in the isolated DBD fragment or in the presence of the TD mutation L344P, which abolishes tetramerization. Misfolding appears to involve intramolecular DBD-DBD association within a single tetrameric molecule. This association is promoted by destabilization of DBD (caused by mutation or elevated temperature) and by the high local DBD concentration enforced by tetramerization of TD. Disrupting the nonnative DBD-DBD interaction or transiently inhibiting tetramerization and allowing p53 to fold as a monomer may be potential strategies for pharmacological intervention in cancer.     

Conformational transition between four and five-stranded phenylalanine zippers determined by a local packing interaction

Alpha-helical coiled coils play a crucial role in mediating specific protein-protein interactions. However, the rules and mechanisms that govern helix-helix association in coiled coils remain incompletely understood. Here we have engineered a seven heptad "Phe-zipper" protein (Phe-14) with phenylalanine residues at all 14 hydrophobic a and d positions, and generated a further variant (Phe-14(M)) in which a single core Phe residue is substituted with Met. Phe-14 forms a discrete alpha-helical pentamer in aqueous solution, while Phe-14(M) folds into a tetrameric helical structure. X-ray crystal structures reveal that in both the tetramer and the pentamer the a and d side-chains interlock in a classical knobs-into-holes packing to produce parallel coiled-coil structures enclosing large tubular cavities. However, the presence of the Met residue in the apolar interface of the tetramer markedly alters its local coiled-coil conformation and superhelical geometry. Thus, short-range interactions involving the Met side-chain serve to preferentially select for tetramer formation, either by inhibiting a nucleation step essential for pentamer folding or by abrogating an intermediate required to form the pentamer. Although specific trigger sequences have not been clearly identified in dimeric coiled coils, higher-order coiled coils, as well as other oligomeric multi-protein complexes, may require such sequences to nucleate and direct their assembly.     

A parallel coiled-coil tetramer with offset helices

Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between alpha helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of alpha-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valine-containing mutant forms a discrete alpha-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 A resolution crystal structure of the tetramer reveals a parallel four-stranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.     

Solvent-exposed residues located in the beta-sheet modulate the stability of the tetramerization domain of p53--a structural and combinatorial approach

The role of hydrophobic amino acids in the formation of hydrophobic cores as one of the major driving forces in protein folding has been extensively studied. However, the implication of neutral solvent-exposed amino acids is less clear and available information is scarce. We have used a combinatorial approach to study the structural relevance of three solvent-exposed residues (Tyr(327), Thr(329), and Gln(331)) located in thebeta-sheet of the tetramerization domain of the tumor suppressor p53 (p53TD). A conformationally defined peptide library was designed where these three positions were randomized. The library was screened for tetramer stability. A set of p53TD mutants containing putative stabilizing or destabilizing residue combinations was synthesized for a thermodynamic characterization. Unfolding experiments showed a wide range of stabilities, with T(m) values between 27 and 83 degrees C. Wild type p53TD and some highly destabilized and stabilized mutants were further characterized. Thermodynamic and biophysical data indicated that these proteins were folded tetramers, with the same overall structure, in equilibrium with unfolded monomers. An NMR study confirmed that the main structural features of p53TD are conserved in all the mutants analyzed. The thermodynamic stability of the different p53TD mutants showed a strong correlation with parameters that favor formation and stabilization of the beta-sheet. We propose that stabilization through hydrophobic interactions of key secondary structure elements might be the underlying mechanism for the strong influence of solvent-exposed residues in the stability of p53TD.     

Solution structure of a small protein containing a fluorinated side chain in the core

We report the first high-resolution structure for a protein containing a fluorinated side chain. Recently we carried out a systematic evaluation of phenylalanine to pentafluorophenylalanine (Phe --> F(5)-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F(5)-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than either aryl-aryl or perfluoroaryl-perfluoroaryl interactions, and because perfluoroaryl units are more hydrophobic than are analogous aryl units. Only one mutation, Phe10 --> F(5)-Phe, was found to provide enhanced tertiary structural stability relative to the native core (by approximately 1 kcal/mol, according to guanidinium chloride denaturation studies). The NMR structure of this mutant, described here, reveals very little variation in backbone conformation or side chain packing relative to the wild type. Thus, although Phe --> F(5)-Phe mutations offer the possibility of greater tertiary structural stability from side chain-side chain attraction and/or side chain desolvation, the constraints associated with the native c-VHP fold apparently prevent the modified polypeptide from taking advantage of this possibility. Our findings are important because they complement several studies that have shown that fluorination of saturated side chain carbon atoms can provide enhanced conformational stability.     

A stable human p53 heterotetramer based on constructive charge interactions within the tetramerization domain

The human p53 tetramerization domain (called p53tet; residues 325-355) spontaneously forms a dimer of dimers in solution. Hydrophobic interactions play a major role in stabilizing the p53 tetramer. However, the distinctive arrangement of charged residues at the dimer-dimer interface suggests that they also contribute to tetramer stability. Charge-reversal mutations at positions 343, 346, and 351 within the dimer-dimer interface were thus introduced into p53tet constructs and shown to result in the selective formation of a stable heterotetramer composed of homodimers. More precisely, mutants p53tet-E343K/E346K and p53tet-K351E preferentially associated with each other, but not with wild-type p53tet, to form a heterodimeric tetramer with enhanced thermal stability relative to either of the two components in isolation. The p53tet-E343K/E346K mutant alone assembled into a weakly stable tetramer in solution, whereas p53tet-K351E existed only as a dimer. Moreover, these mutants did not form heterocomplexes with wild-type p53tet, illustrating the specificity of the ionic interactions that form the novel heterotetramer. This study demonstrates the dramatic importance of ionic interactions in altering the stability of the p53 tetramer and in selectively creating heterotetramers of this protein scaffold.     

Hydrophobic side-chain size is a determinant of the three-dimensional structure of the p53 oligomerization domain.

The p53 tumor suppressor oligomerization domain, a dimer of two primary dimers, is an independently folding domain whose subunits consist of a beta-strand, a tight turn and an alpha-helix. To evaluate the effect of hydrophobic side-chains on three-dimensional structure, we substituted residues Phe341 and Leu344 in the alpha-helix with other hydrophobic amino acids. Substitutions that resulted in residue 341 having a smaller side-chain than residue 344 switched the stoichiometry of the domain from tetrameric to dimeric. The three-dimensional structure of one such dimer was determined by multidimensional NMR spectroscopy. When compared with the primary dimer of the wild-type p53 oligomerization domain, the mutant dimer showed a switch in alpha-helical packing from anti-parallel to parallel and rotation of the alpha-helices relative to the beta-strands. Hydrophobic side-chain size is therefore an important determinant of a protein fold.     

Change in oligomerization specificity of the p53 tetramerization domain by hydrophobic amino acid substitutions.

The tumor suppressor function of the wild-type p53 protein is transdominantly inhibited by tumor-derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild-type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild-type p53 subunits into inactive wild-type/mutant hetero-tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor-derived mutant p53, would escape transdominant inhibition. Aided by the known three-dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full-length p53 protein bearing this novel domain formed homo-tetramers and had tumor suppressor function, but did not hetero-oligomerize with tumor-derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain.