Allosteric switching by mutually exclusive folding of protein domains

Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction.     

Alanine Scan of Core Positions in Ubiquitin Reveals Links between Dynamics,Stability, and Function

Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.     

Measuring the stability of partly folded proteins using TMAO

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.     

Natural selection for kinetic stability is a likely origin of correlations between mutational effects on protein energetics and frequencies of amino acid occurrences in sequence alignments

It appears plausible that natural selection constrains, to some extent at least, the stability in many natural proteins. If, during protein evolution, stability fluctuates within a comparatively narrow range, then mutations are expected to be fixed with frequencies that reflect mutational effects on stability. Indeed, we recently reported a robust correlation between the effect of 27 conservative mutations on the thermodynamic stability (unfolding free energy) of Escherichia coli thioredoxin and the frequencies of residues occurrences in sequence alignments. We show here that this correlation likely implies a lower limit to thermodynamic stability of only a few kJ/mol below the unfolding free energy of the wild-type (WT) protein. We suggest, therefore, that the correlation does not reflect natural selection of thermodynamic stability by itself, but of some other factor which is linked to thermodynamic stability for the mutations under study. We propose that this other factor is the kinetic stability of thioredoxin in vivo, since( i) kinetic stability relates to irreversible denaturation, (ii) the rate of irreversible denaturation in a crowded cellular environment (or in a harsh extracellular environment) is probably determined by the rate of unfolding, and (iii) the half-life for unfolding changes in an exponential manner with activation free energy and, consequently, comparatively small free energy effects can have deleterious consequences for kinetic stability. This proposal is supported by the results of a kinetic study of the WT form and the 27 single-mutant variants of E. coli thioredoxin based on the global analyses of chevron plots and equilibrium unfolding profiles determined from double-jump unfolding assays. This kinetic study suggests, furthermore, one of the factors that may contribute to the high activation free energy for unfolding in thioredoxin (required for kinetic stability), namely the energetic optimization of native-state residue environments in regions, which become disrupted in the transition state for unfolding.     

Modular enzyme design: regulation by mutually exclusive protein folding

A regulatory mechanism is introduced whereupon the catalytic activity of a given enzyme is controlled by ligand binding to a receptor domain of choice. A small enzyme (barnase) and a ligand-binding polypeptide (GCN4) are fused so that a simple topological constraint prevents them from existing simultaneously in their folded states. The two domains consequently engage in a thermodynamic tug-of-war in which the more stable domain forces the less stable domain to unfold. In the absence of ligand, the barnase domain is more stable and is therefore folded and active; the GCN4 domain is substantially unstructured. DNA binding induces folding of GCN4, forcibly unfolding and inactivating the barnase domain. Barnase-GCN4 is thus a "natively unfolded" protein that uses ligand binding to switch between partially folded forms. The key characteristics of each parent protein (catalytic efficiency of barnase, DNA binding affinity and sequence specificity of GCN4) are retained in the chimera. Barnase-GCN4 thus defines a modular approach for assembling enzymes with novel sensor capabilities from a variety of catalytic and ligand binding domains.     

A mini-protein designed by removing a module from barnase: molecular modeling and NMR measurements of the conformation.

A globular domain can be decomposed into compact modules consisting of contiguous 10-30 amino acid residues. The correlation between modules and exons observed in different proteins suggests that each module was encoded by an ancestral exon and that modules were combined into globular domains by exon fusion. Barnase is a single domain RNase consisting of 110 amino acid residues and was decomposed into six modules. We designed a mini-protein by removing the second module, M2, from barnase in order to gain an insight into the structural and functional roles of the module. In the molecular modeling of the mini-protein, we evaluated thermodynamic stability and aqueous solubility together with mechanical stability of the model. We chemically synthesized a mini-barnase with (15)N-labeling at 10 residues, whose corresponding residues in barnase are all found in the region around the hydrophobic core. Circular dichroism and NMR measurements revealed that mini-barnase takes a non-random specific conformation that has a similar hydrophobic core structure to that of barnase. This result, that a module could be deleted without altering the structure of core region of barnase, supports the view that modules act as the building blocks of protein design.     

Stability and folding of the protein complexes of barnase.

Native-like complexes of proteins, formed by the association of two complementary fragments comprising the entire sequence of the protein, can be used to gain insight into the stability and folding of the intact protein. We have studied the structural, thermodynamic and kinetic properties of four barnase complexes, with the cleavage site at different positions of the amino-acid chain (CB36, at position 36; CB56, at position 56; CB68, at position 68; and CB79, at position 79). The four barnase complexes have native-like structure as shown by fluorescence, far-and near-UV CD, size-exclusion chromatography and NMR. The NMR characterization indicated that the structural changes were mainly located in regions close to the cleavage site. The main core of the protein was fully formed and the overall structure was similar to that of intact barnase. The thermal and chemical denaturation showed that all complexes were substantially destabilized. CB56 displayed two denaturation transitions, probably because of the presence of partially folded conformations around the cleavage site. The rate constant for the association/folding of fragments decreased with the decreasing length of the C-terminal fragment. Thus, the larger the fragment (and, consequently, the larger the amount of residual native-like structure), the faster the association. These findings are consistent with the proposed model of barnase folding.     

Topological thermal instability and length of proteins

We present an analysis of the effects of global topology on the structural stability of folded proteins in thermal equilibrium with a heat bath. For a large class of single domain proteins, we computed the harmonic spectrum within the Gaussian Network Model (GNM) and determined their spectral dimension, a parameter describing the low frequency behavior of the density of modes. We found a surprisingly strong correlation between the spectral dimension and the number of amino acids in the protein. Considering that larger spectral dimension values relate to more topologically compact folded states, our results indicate that, for a given temperature and length of protein, the folded structure corresponds to a less compact folding, one compatible with thermodynamic stability.     

The influence of intrinsic folding mechanism of an unfolded protein on the coupled folding-binding process during target recognition

Intrinsically disordered proteins (IDPs) are extensively involved in dynamic signaling processes which require a high association rate and a high dissociation rate for rapid binding/unbinding events and at the same time a sufficient high affinity for specific recognition. Although the coupled folding-binding processes of IDPs have been extensively studied, it is still impossible to predict whether an unfolded protein is suitable for molecular signaling via coupled folding-binding. In this work, we studied the interplay between intrinsic folding mechanisms and coupled folding-binding process for unfolded proteins through molecular dynamics simulations. We first studied the folding process of three representative IDPs with different folded structures, that is, c-Myb, AF9, and E3 rRNase. We found the folding free energy landscapes of IDPs are downhill or show low barriers. To further study the influence of intrinsic folding mechanism on the binding process, we modulated the folding mechanism of barnase via circular permutation and simulated the coupled folding-binding process between unfolded barnase permutant and folded barstar. Although folding of barnase was coupled to target binding, the binding kinetics was significantly affected by the intrinsic folding free energy barrier, where reducing the folding free energy barrier enhances binding rate up to two orders of magnitude. This accelerating effect is different from previous results which reflect the effect of structure flexibility on binding kinetics. Our results suggest that coupling the folding of an unfolded protein with no/low folding free energy barrier with its target binding may provide a way to achieve high specificity and rapid binding/unbinding kinetics simultaneously.     

Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities

"Host-guest" studies of the B1 domain from Streptococcal protein G have been used previously to establish a thermodynamic scale for the beta-sheet-forming propensities of the 20 common amino acids. To investigate the contribution of side chain conformational entropy to the relative stabilities of B1 domain mutants, we have determined the dynamics of side chain methyl groups in 10 of the 20 mutants used in a previous study. Deuterium relaxation rates were measured using two-dimensional NMR techniques for 13CH2D groups. Analysis of the relaxation data using the Lipari-Szabo model-free formalism showed that mutations introduced at the guest position caused small but statistically significant changes in the methyl group dynamics. In addition, there was a low level of covariation of the Lipari-Szabo order parameters among the 10 mutants. The variations in conformational free energy estimated from the order parameters were comparable in magnitude to the variations in global stability of the 10 mutants but did not correlate with the global stability of the domain or with the structural properties of the guest amino acids. The data support the view that conformational entropy in the folded state is one of many factors that can influence the folding thermodynamics of proteins.     

An irregular beta-bulge common to a group of bacterial RNases is an important determinant of stability and function in barnase

Single amino acid residue substitutions rarely destroy the structural integrity of proteins. Substitution of glycine residues, however, is among the few sorts of alterations that can have such an effect. Here, we seek to understand what accounts for the extreme functional impairment of the bacterial ribonuclease barnase upon substitution of Gly52 or Gly53. We find that inactivation is caused by overall disruption of the folded state that manifests itself in three ways: (1) dramatically reduced stability (by 5.2 to 8.4 kcal mol-1 for mutants showing inactivation in vivo); (2) progressive loss of folded-state activity with increasing temperature, indicating a less well formed fold; and (3) substantial proteolytic degradation of mutant enzymes in vivo. Examination of two deletion mutants, missing either Gly53 or Asp54, shows that the irregular beta-bulge formed by these two residues is of vital importance to the structural integrity of barnase. The parallel behaviour of mutants carrying replacements of either of the two glycine residues therefore appears to arise from a common mechanism: disruption of local structure at the beta-bulge. The importance of this structural element to the function of barnase raises the question of whether it may be present in other RNases. The Streptomyces enzymes RNase Sa and RNase St differ considerably from barnase in both sequence and structure, yet both show significant sequence similarity to barnase over a region beginning at Gly53. Structural comparison indicates that the Streptomyces enzymes do have the barnase-like irregular beta-bulge, making this an important characteristic feature of a group of bacterial ribonucleases. The sensitivity of this feature demonstrates that detailed aspects of local structure may have a major role in determining the overall structural and functional properties of an enzyme, even where no explanation for this role is readily apparent. If this is a general characteristic of the structure-function relationship, it may pose a formidable obstacle to the de novo design of new enzymes.     

A single disulfide bond restores thermodynamic and proteolytic stability to an extensively mutated protein

The potential for engineering stable proteins with multiple amino acid substitutions was explored. Eleven lysine, five methionine, two tryptophan, one glycine, and three threonine substitutions were simultaneously made in barley chymotrypsin inhibitor-2 (CI-2) to substantially improve the essential amino acid content of the protein. These substitutions were chosen based on the three-dimensional structure of CI-2 and an alignment of homologous sequences. The initial engineered protein folded into a wild-type-like structure, but had a free energy of unfolding of only 2.2 kcal/mol, considerably less than the wild-type value of 7.5 kcal/mol. Restoration of the lysine mutation at position 67 to the wild-type arginine increased the free energy of unfolding to 3.1 kcal/mol. Subsequent cysteine substitutions at positions 22 and 82 resulted in disulfide bond formation and a protein with nearly wild-type thermodynamic stability (7.0 kcal/mol). None of the engineered proteins retained inhibitory activity against chymotrypsin or elastase, and all had substantially reduced inhibitory activity against subtilisin. The proteolytic stabilities of the proteins correlated with their thermodynamic stabilities. Reduction of the disulfide bond resulted in substantial loss of both thermodynamic and proteolytic stabilities, confirming that the disulfide bond, and not merely the cysteine substitutions, was responsible for the increased stability. We conclude that it is possible to replace over a third of the residues in CI-2 with minimal disruption of stability and structural integrity.     

Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains

Ubiquilin/PLIC proteins belong to the family of UBL-UBA proteins implicated in the regulation of the ubiquitin-dependent proteasomal degradation of cellular proteins. A human presenilin-interacting protein, ubiquilin-1, has been suggested as potential therapeutic target for treating Huntington's disease. Ubiquilin's interactions with mono- and polyubiquitins are mediated by its UBA domain, which is one of the tightest ubiquitin binders among known ubiquitin-binding domains. Here we report the three-dimensional structure of the UBA domain of ubiquilin-1 (UQ1-UBA) free in solution and in complex with ubiquitin. UQ1-UBA forms a compact three-helix bundle structurally similar to other known UBAs, and binds to the hydrophobic patch on ubiquitin with a K_d of 20 μM. To gain structural insights into UQ1-UBA's interactions with polyubiquitin chains, we have mapped the binding interface between UQ1-UBA and Lys48- and Lys63-linked di-ubiquitins and characterized the strength of UQ1-UBA binding to these chains. Our NMR data show that UQ1-UBA interacts with the individual ubiquitin units in both chains in a mode similar to its interaction with mono-ubiquitin, although with an improved binding affinity for the chains. Our results indicate that, in contrast to UBA2 of hHR23A that has strong binding preference for Lys48-linked chains, UQ1-UBA shows little or no binding selectivity toward a particular chain linkage or between the two ubiquitin moieties in the same chain. The structural data obtained in this study provide insights into the possible structural reasons for the diversity of polyubiquitin chain recognition by UBA domains.     

Pathway and stability of protein folding.

We describe an experimental approach to the problem of protein folding and stability which measures interaction energies and maps structures of intermediates and transition states during the folding pathway. The strategy is based on two steps. First, protein engineering is used to remove interactions that stabilize defined positions in barnase, the RNAse from Bacillus amyloliquefaciens. The consequent changes in stability are measured from the changes in free energy of unfolding of the protein. Second, each mutation is used as a probe of the structure around the wild-type side chain during the folding process. Kinetic measurements are made on the folding and unfolding of wild-type and mutant proteins. The kinetic and thermodynamic data are combined and analysed to show the role of individual side chains in the stabilization of the folded, transition and intermediate states of the protein. The protein engineering experiments are corroborated by nuclear magnetic resonance studies of hydrogen exchange during the folding process. Folding is a multiphasic process in which alpha-helices and beta-sheet are formed relatively early. Formation of the hydrophobic core by docking helix and sheet is (partly) rate determining. The final steps involve the forming of loops and the capping of the N-termini of helices.     

Strength and co-operativity of contributions of surface salt bridges to protein stability

Many of the interactions that stabilize proteins are co-operative and cannot be reduced to a sum of pairwise interactions. Such interactions may be analysed by protein engineering methods using multiple thermodynamic cycles comprising wild-type protein and all combinations of mutants in the interacting residues. There is a triad of charged residues on the surface of barnase, comprising residues Asp8, Asp12 and Arg110, that interact by forming two exposed salt bridges. The three residues have been mutated to alanine to give all the single, double and triple mutants. The free energies of unfolding of wild-type and the seven mutant proteins have been determined and the results analysed to give the contributions of the residues in the two salt bridges to protein stability. It is possible to isolate the energies of forming the salt bridges relative to the solvation of the separated ions by water. In the intact triad, the apparent contribution to the stabilization energy of the protein of the salt bridge between Asp12 and Arg110 is -1.25 kcal mol-1, whereas that of the salt bridge between Asp8 with Arg110 is -0.98 kcal mol-1. The strengths of the two salt bridges are coupled: the energy of each is reduced by 0.77 kcal mol-1 when the other is absent. The salt-linked triad, relative to alanine residues at the same positions, does not contribute to the stability of the protein since the favourable interactions of the salt bridges are more than offset by other electrostatic and non-electrostatic energy terms. Salt-linked triads occur in other proteins, for example, haemoglobin, where the energy of only the salt-bridge term is important and so the coupling of salt bridges could be of general importance to the stability and function of proteins.     

Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment

The thermodynamic and kinetic stabilities of the eye lens family of betagamma-crystallins are important factors in the etiology of senile cataract. They control the chance of proteins unfolding, which can lead to aggregation and loss of transparency. betaB2-Crystallin orthologs are of low stability and comprise two typical betagamma-crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded beta-hairpins. Using high-temperature (500 K) molecular dynamics simulations with explicit solvent on the N-terminal domain of rodent betaB2-crystallin, we have identified in silico local flexibility in this folded beta-hairpin. We have shown in vitro using two-domain human betaB2-crystallin that replacement of this cysteine with a more usual aromatic residue (phenylalanine) results in a gain in conformational stability and a reduction in the rate of unfolding. We have used principal components analysis to visualize and cluster the coordinates from eight separate simulated unfolding trajectories of both the wild-type and the C50F mutant N-terminal domains. These data, representing fluctuations around the native well, show that although the mutant and wild-type appear to behave similarly over the early time period, the wild type appears to explore a different region of conformational space. It is proposed that the advantage of having this low-stability cysteine may be correlated with a subunit-exchange mechanism that allows betaB2-crystallin to interact with a range of other beta-crystallin subunits.     

Effect of Interdomain Linker Length on an Antagonistic Folding-Unfolding Equilibrium between Two Protein Domains

Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (ΔG_X). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. ΔG_X values are determined by measuring the extent to which Co²⁺ binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that ΔG_X increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a three-dimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site.     

Ubiquitinated Proteins Activate the Proteasomal ATPases by Binding to Usp14 or Uch37 Homologs

Degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis, but it is unclear how the proteasomal ATPases are regulated and how proteolysis, substrate deubiquitination, degradation, and ATP hydrolysis are coordinated. Polyubiquitinated proteins were shown to stimulate ATP hydrolysis by purified proteasomes, but only if the proteins contain a loosely folded domain. If they were not ubiquitinated, such proteins did not increase ATPase activity. However, they did so upon addition of ubiquitin aldehyde, which mimics the ubiquitin chain and binds to 26 S-associated deubiquitinating enzymes (DUBs): in yeast to Ubp6, which is essential for the ATPase activation, and in mammalian 26 S to the Ubp6 homolog, Usp14, and Uch37. Occupancy of either DUB by a ubiquitin conjugate leads to ATPase stimulation, thereby coupling deubiquitination and ATP hydrolysis. Thus, ubiquitinated loosely folded proteins, after becoming bound to the 26 S, interact with Ubp6/Usp14 or Uch37 to activate ATP hydrolysis and enhance their own destruction.     

Mutant p62/SQSTM1 UBA domains linked to Paget’s disease of bone differ in their abilities to function as stabilization signals

We show that the ubiquitin-associated domain (UBA) of human p62/sequestosome-1 (SQSTM1) can delay degradation of proteasome substrates in yeast. Taking advantage of naturally occurring mutant UBA domains that are linked to Paget’s disease of bone (PDB), we found that three of the four mutant UBA domains tested in this study were able to inhibit proteasomal degradation, albeit not to the same extent as the wild-type domain. Interestingly, the stability measured as the fraction of folded protein, and not the ubiquitin binding properties, of the PDB-associated UBA domains correlated with their protective effects. These data suggest that the protective effect of UBA domains depends on their structural integrity rather than ubiquitin binding capabilities.