Search

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3Kα and PI3Kγ require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3Kα and PI3Kγ, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3Kα interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3Kα bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3Kα and PI3Kγ that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 A X-ray crystal structure of the PI3Kα-RBD, which aligns well to full-length PI3Kα. Structural differences between the PI3Kα and PI3Kγ RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction.

Pathways of ubiquitin-like (UBL) molecule transfer regulate a myriad of cellular cascades. Here, we report a high-throughput assay that correlates catalytic human NEDD8 transfer to bacterial survival. The assay was utilized to screen mutant NEDD8 and NAE (NEDD8-activating enzyme) libraries to engineer a more stable NEDD8 and redesign the NEDD8-NAE interface. This approach will be useful in understanding the specificities underlying UBL pathways.

The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: 1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, 2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild type complex and 3) the mutation should not be predicted to significantly destabilize the monomers. The Rosetta energy function emphasizes short-range interactions: steric repulsion, Van der Waals forces, hydrogen bonding, and an implicit solvation model that penalizes placing atoms adjacent to polar groups. The performance of the computational protocol was experimentally tested on two separate protein complexes; Gαi1 from the heterotrimeric G-protein system bound to the RGS14 GoLoco motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. 12 single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. 9 of the 12 mutations successfully increased binding affinity with 5 of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the 8 mutations identified, 5 were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities.

Recent efforts to design de novo or redesign the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol RosettaDesign to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal/mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we have designed and characterized a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations; one mutant gains more than 5 kcal/mol of stability over the wild-type protein with only four amino acid changes. We explore the relationship between force-field smoothing and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using modified force-fields, and both fixed and flexible backbone sampling protocols.

The structurally defined ubiquitin-like homology fold (UBL) can engage in several unique protein–protein interactions and many of these complexes have been characterized with high-resolution techniques. Using Rosetta's structural classification tools, we have created the Ubiquitin Structural Relational Database (UbSRD), an SQL database of features for all 509 UBL-containing structures in the PDB, allowing users to browse these structures by protein–protein interaction and providing a platform for quantitative analysis of structural features. We used UbSRD to define the recognition features of ubiquitin (UBQ) and SUMO observed in the PDB and the orientation of the UBQ tail while interacting with certain types of proteins. While some of the interaction surfaces on UBQ and SUMO overlap, each molecule has distinct features that aid in molecular discrimination. Additionally, we find that the UBQ tail is malleable and can adopt a variety of conformations upon binding. UbSRD is accessible as an online resource at rosettadesign.med.unc.edu/ubsrd.

We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mis-translated and accumulates at high levels in Escherichia coli. We used circular dichroism, size-exclusion chromatography, small-angle X-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting C-terminal fragment (CFr) protein adopts a compact, extremely stable, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery. The rarity of correspondingly stable fragments in natural proteins coupled with the observation that high quality ribosome binding sites are found to occur within E. coli protein-coding regions significantly less often than expected by random chance implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mis-translated CFr sub-domains to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of protein sub-structures.

A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7 kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.

Protein –protein docking algorithms provide a means to elucidate structural details for presently unknown complexes. Here, we present and evaluate a new method to predict protein –protein complexes from the coordinates of the unbound monomer components. The method employs a low-resolution, rigid-body, Monte Carlo search followed by simultaneous optimization of backbone displacement and side-chain conformations using Monte Carlo minimization. Up to 10 5 independent simulations are carried out, and the resulting “decoys” are ranked using an energy function dominated by van der Waals interactions, an implicit solvation model, and an orientation-dependent hydrogen bonding potential. Top-ranking decoys are clustered to select the final predictions. Small-perturbation studies reveal the formation of binding funnels in 42 of 54 cases using coordinates derived from the bound complexes and in 32 of 54 cases using independently determined coordinates of one or both monomers. Experimental binding affinities correlate with the calculated score function and explain the predictive success or failure of many targets. Global searches using one or both unbound components predict at least 25% of the native residue –residue contacts in 28 of the 32 cases where binding funnels exist. The results suggest that the method may soon be useful for generating models of biologically important complexes from the structures of the isolated components, but they also highlight the challenges that must be met to achieve consistent and accurate prediction of protein –protein interactions. q 2003 Elsevier Ltd. All rights reserved

The rational design of loops and turns is a key step towards creating proteins with new functions. We used a computational design procedure to create new backbone conformations in the second turn of protein L. The Protein Data Bank was searched for alternative turn conformations, and sequences optimal for these turns in the context of protein L were identified using a Monte Carlo search procedure and an energy function that favors close packing. Two variants containing 12 and 14 mutations were found to be as stable as wild-type protein L. The crystal structure of one of the variants has been solved at a resolution of 1.9 A, and the backbone conformation in the second turn is remarkably close to that of the in silico model (1.1 A RMSD) while it differs significantly from that of wild-type protein L (the turn residues are displaced by an average of 7.2 A). The folding rates of the redesigned proteins are greater than that of the wild-type protein and in contrast to wild-type protein L the second beta-turn appears to be formed at the rate limiting step in folding.

The N-terminal domain of the ribosomal protein L9 forms a split βαβ structure with a long C-terminal helix. The folding transitions of a 56 residue version of this protein have previously been characterized, here we report the results of a study of a truncation mutant corresponding to residues 1–51. The 51 residue protein adopts the same fold as the 56 residue protein as judged by CD and two-dimensional NMR, but it is less stable as judged by chemical and thermal denaturation experiments. Studies with synthetic peptides demonstrate that the C-terminal helix of the 51 residue version has very little propensity to fold in isolation in contrast to the C-terminal helix of the 56 residue variant. The folding rates of the two proteins, as measured by stopped-flow fluorescence, are essentially identical, indicating that formation of local structure in the C-terminal helix is not involved in the rate-limiting step of folding.

The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been measured at temperatures between 7 and 85°C and between 0 and 6 M guanidine deuterium chloride. Stopped-flow fluorescence was used to measure rates below 55°C and NMR lineshape analysis was used above 55°C. The amplitudes and rate profiles of the stopped-flow fluorescence experiments are consistent with a two-state folding mechanism, and plots of ln ( k ) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were fit simultaneously to the simple model k = D exp(−Δ G ‡ / RT ) where Δ G ‡ represents the change in apparent free energy between the transition state and the folded or unfolded state and D represents the maximum possible folding speed. Δ G ‡ is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to model stability changes with temperature. Approximately 60% of the surface area buried upon folding is buried in the transition state as evidenced by changes in the heat capacity and m value between the unfolded state and the transition state. The equilibrium thermodynamic parameters, Δ C p °, m and Δ G °, all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-state. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius temperature dependence typical of globular proteins. When the folding rates are examined along constant Δ G °/ T contours they display an Arrhenius temperature dependence with a slope of −8600 K. This indicates that for this system, the non-Arrhenius temperature dependence of folding can be accounted for by the anomalous temperature dependence of the interactions which stabilize proteins.

The peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus is stably folded, despite its short sequence of only 43 amino acid residues. A 41 residue peptide derived from this domain, psbd41, undergoes a cooperative thermal unfolding transition with a t m of 54°C. This three-helix protein is monomeric as judged by ultracentrifugation and concentration-dependent CD measurements. Peptides corresponding to the individual helices are largely unstructured both alone and in combination, indicating that the unusual stability of this protein does not arise solely from unusually stable α-helices. Chemical denaturation by guanidine hydrochloride is also cooperative with a Δ G H2O of 3.1 kcal mol −1 at pH 8.0 and 25°C. The chemical denaturation is broad with an m -value of 760 cal mol −1 M −1 . psbd41 contains a buried aspartate residue at position 34 that may provide stability and specificity to the fold. A mutant peptide, psbd41Asn was synthesized in which the buried aspartate residue was mutated to asparagine. This peptide still folds cooperatively and it is monomeric, but is much less thermostable than the wild-type with a t m of only 31°C. Chemical denaturations at 4°C give an m -value of 740 cal mol −1 M −1 , similar to the wild-type, but the stability Δ G H2O is only 1.4 kcal mol −1 . Both the wild-type and the mutant unfold at extremes of pH, but at 4°C psbd41Asn is folded over a narrower pH range than the wild-type. Although the mutant unfolds cooperatively by thermal and by chemical denaturation, its NMR spectrum is significantly broader than that of the wild-type and it binds ANS. These results show that Asp34 is vital for the stability and specificity of this structure, the second smallest natural sequence known to fold in the absence of disulfide bonds or metal or ligand-binding sites.

The ribosomal protein L9 has an unusual structure comprising two compact globular domains connected by a 34 residue alpha-helix. The middle 17 residues of the helix are exposed to solvent while the first seven pack against and form part of the N-terminal domain, and the last ten form part of the C-terminal domain. Here we report results which show that a peptide corresponding to the central helix of L9 is monomeric in aqueous solution and85% helical at 1 degrees C and 68(+/-7)% helical at 25 degrees C. This is considerably more helical than any other protein fragment studied to date. Another peptide corresponding to the middle 17 residues of the helix is monomeric and is 41(+/-4)% helical at 1 degrees C. Because the central helix has high intrinsic stability the globular N and C-terminal domains will likely be stabilized by their interactions with the helix. Therefore, the stability of the two terminal domains should not be completely independent because both domains gain stability from a shared structural element, the central helix. Also, the ability of the central helix to form native-like structure in isolation highlights a potential role for the helix in the early stages of the folding process.