Erythropoietin receptor activation by a ligand-induced conformation change.

Erythropoietin and other cytokine receptors are thought to be activated through hormone-induced dimerization and autophosphorylation of JAK kinases associated with the receptor intracellular domains. An in vivo protein fragment complementation assay was used to obtain evidence for an alternative mechanism in which unliganded erythropoietin receptor dimers exist in a conformation that prevents activation of JAK2 but then undergo a ligand-induced conformation change that allows JAK2 to be activated. These results are consistent with crystallographic evidence of distinct dimeric configurations for unliganded and ligand-bound forms of the erythropoietin receptor.

Solution structure of FKBP, a rotamase enzyme and receptor for FK506 and rapamycin.

Immunophilins, when complexed to immunosuppressive ligands, appear to inhibit signal transduction pathways that result in exocytosis and transcription. The solution structure of one of these, the human FK506 and rapamycin binding protein (FKBP), has been determined by nuclear magnetic resonance (NMR). FKBP has a previously unobserved antiparallel beta-sheet folding topology that results in a novel loop crossing and produces a large cavity lined by a conserved array of aromatic residues; this cavity serves as the rotamase active site and drug-binding pocket. There are other significant structural features (such as a protruding positively charged loop and an apparently flexible loop) that may be involved in the biological activity of FKBP.

The transition state of the ras binding domain of Raf is structurally polarized based on Phi-values but is energetically diffuse.

The ras binding domain (RBD) of the Ser/Thr kinase c-Raf/Raf-1 spans 78 residues and adopts a structure characteristic of the beta-grasp ubiquitin-like topology. Recently, the primary sequence of Raf RBD has been nearly exhaustively mutated experimentally by insertion of stretches of degenerate codons, which revealed sequence conservation and hydrophobic core organization similar to that found in an alignment of beta-grasp ubiquitin-like proteins. These results now allow us to examine the relationship between sequence conservation and the folding process, particularly viewed through the analysis of transition state (TS) structure. Specifically, we present herein a protein engineering study combining classic truncation (Ala/Gly) and atypical mutants to predict folding TS ensemble properties. Based on classical Phi-value analysis, Raf RBD TS structure is particularly polarized around the N-terminal beta-hairpin. However, all residues constituting the inner layer of the hydrophobic core are involved in TS stabilization, although they are clearly found in a less native-like environment. The TS structure can also be probed by a direct measure of its destabilization upon mutation, DeltaDeltaG(U-++). Viewed through this analysis, Raf RBD TS is a more diffuse structure, in which all residues of the hydrophobic core including beta-strands 1, 2, 3 and 5 and the major alpha-helix play similar roles in TS stabilization. In addition, Phi-values and DeltaDeltaG(U-++) reveal striking similarities in the TS of Raf RBD and ubiquitin, a structural analogue displaying insignificant sequence identity (<12%). However, ubiquitin TS appears more denatured-like and polarized around the N-terminal beta-hairpin. We suggest that analysis of Phi-values should also consider the direct impact of mutations on differences in free energy between the unfolded and TS (DeltaDeltaG(U-++)) to ensure that the description of TS properties is accurate. Finally, the impact of these findings on the modeling of protein folding is discussed.

Massive sequence perturbation of the Raf ras binding domain reveals relationships between sequence conservation, secondary structure propensity, hydrophobic core organization and stability.

The contributions of specific residues to the delicate balance between function, stability and folding rates could be determined, in part by [corrected] comparing the sequences of structures having identical folds, but insignificant sequence homology. Recently, we have devised an experimental strategy to thoroughly explore residue substitutions consistent with a specific class of structure. Using this approach, the amino acids tolerated at virtually all residues of the c-Raf/Raf1 ras binding domain (Raf RBD), an exemplar of the common beta-grasp ubiquitin-like topology, were obtained and used to define the sequence determinants of this fold. Herein, we present analyses suggesting that more subtle sequence selection pressure, including propensity for secondary structure, the hydrophobic core organization and charge distribution are imposed on the Raf RBD sequence. Secondly, using the Gibbs free energies (DeltaG(F-U)) obtained for 51 mutants of Raf RBD, we demonstrate a strong correlation between amino acid conservation and the destabilization induced by truncating mutants. In addition, four mutants are shown to significantly stabilize Raf RBD native structure. Two of these mutations, including the well-studied R89L, are known to severely compromise binding affinity for ras. Another stabilized mutant consisted of a deletion of amino acid residues E104-K106. This deletion naturally occurs in the homologues a-Raf and b-Raf and could indicate functional divergence. Finally, the combination of mutations affecting five of 78 residues of Raf RBD results in stabilization of the structure by approximately 12 kJ mol(-1) (DeltaG(F-U) is -22 and -34 kJ mol(-1) for wt and mutant, respectively). The sequence perturbation approach combined with sequence/structure analysis of the ubiquitin-like fold provide a basis for the identification of sequence-specific requirements for function, stability and folding rate of the Raf RBD and structural analogues, highlighting the utility of conservation profiles as predictive tools of structural organization.

Exploring protein interactions by interaction-induced folding of proteins from complementary peptide fragments.

The study of protein--protein interactions is central to understanding the chemical machinery that makes up the living cell. Until recently, facile methods to study these processes in intact, living cells have not existed. Furthermore, the assignment of function to novel proteins relies on demonstrating interactions of these proteins with proteins of known function. This review describes an experimental strategy, devised to study protein--protein interactions in any intact living cells based on protein-fragment complementation assays. Applications to quantitative analysis of interactions, allosteric processes and cDNA library screening are discussed. Recently, the feasibility of employing this strategy in genome-wide biochemical pathway mapping efforts has been demonstrated.

Direct visualization of protein interactions in plant cells.

The protein NPR1/NIM1 is required for the induction of systemic acquired resistance (SAR) in plants and has been shown to interact with members of the TGA/OBF family of basic leucine zipper (bZIP) transcription factors. However, to date, there is no method available to monitor such interactions in plant cells. We report here an in vivo protein fragment complementation assay (PCA), based on association of reconstituted murine dihydrofolate reductase (mDHFR) with a fluorescent probe to detect protein-protein interaction in planta. We demonstrate that the interaction between Arabidopsis NPR1/NIM1 and the bZIP factor TGA2 is induced by the regulators of SAR, salicylic acid (SA), and its analog 2,6-dichloroisonicotinic acid (INA) with distinct species-specific responses. Furthermore, the induced interaction is localized predominantly in the nucleus. Protein fragment complementation assays could be of value to agricultural research by providing a system for high-throughput biochemical pathway mapping and for screening of small molecules that modulate protein interactions.

An in vivo library-versus-library selection of optimized protein-protein interactions.

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble.

Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-flanking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospecificity by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospecific with a K(D) of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships.

Chemical biology beyond binary codes.

The cellular chemical machinery controlling all living processes is mediated by macroassemblies of enzymes and nucleic acids. These complexes form, break up in different orders and reorganize in ways that result in the chain of events that we call a biochemical 'pathway'. However, all of the methods available to study protein interactions in living cells allow only the observation or recreation of binary complexes. Two recent reports and earlier studies describe the use of chemical 'dimerizers' that can dynamically induce assembly or disassemby of protein complexes. The dimerizer tools create the opportunity to understand the 'real' biology of macromolecular assembly.

Exhaustive enumeration of protein conformations using experimental restraints.

We present an efficient new algorithm that enumerates all possible conformations of a protein that satisfy a given set of distance restraints. Rapid growth of all possible self-avoiding conformations on the diamond lattice provides construction of alpha-carbon representations of a protein fold. We investigated the dependence of the number of conformations on pairwise distance restraints for the proteins crambin, pancreatic trypsin inhibitor, and ubiquitin. Knowledge of between one and two contacts per monomer is shown to be sufficient to restrict the number of candidate structures to approximately 1,000 conformations. Pairwise RMS deviations of atomic position comparisons between pairs of these 1,000 structures revealed that these conformations can be grouped into about 25 families of structures. These results suggest a new approach to assessing alternative protein folds given a very limited number of distance restraints. Such restraints are available from several experimental techniques such as NMR, NOESY, energy transfer fluorescence spectroscopy, and crosslinking experiments. This work focuses on exhaustive enumeration of protein structures with emphasis on the possible use of NOESY-determined distance restraints.

All-atom empirical potential for molecular modeling and dynamics studies of proteins.

New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent-solvent, solvent-solute, and solute-solute interactions. Optimization of the internal parameters used experimental gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the atomic charges, were determined by fitting ab initio interaction energies and geometries of complexes between water and model compounds that represented the backbone and the various side chains. In addition, dipole moments, experimental heats and free energies of vaporization, solvation and sublimation, molecular volumes, and crystal pressures and structures were used in the optimization. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in crystals. A detailed analysis of the relationship between the alanine dipeptide potential energy surface and calculated protein φ, χ angles was made and used in optimizing the peptide group torsional parameters. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in solution and in crystals. Extensive comparisons between molecular dynamics simulations and experimental data for polypeptides and proteins were performed for both structural and dynamic properties. Energy minimization and dynamics simulations for crystals demonstrate that the latter are needed to obtain meaningful comparisons with experimental crystal structures. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of molecules of biological interest.

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo.

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Galpha(s) protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive beta-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.

Massive sequence perturbation of a small protein.

Most protein topologies rarely occur in nature, thus limiting our ability to extract sequence information that could be used to predict structure, function, and evolutionary constraints on protein folds. In principle, the sequence diversity explored by a given protein topology could be expanded by introducing sequence perturbations and selecting variant proteins that fold correctly. However, our capacity to explore sequence space is intrinsically limited by the enormous number of sequences generated from the 20 amino acids and the limited number of variants likely to fold. Here we sought to test whether the sequence space for naturally existing proteins can be explored by simple, sequential degeneration of a complete set of short sequence segments of a model protein, without long-range covariation. Using the Raf ras binding domain as a model of a small protein capable of autonomous folding, we degenerated 72 of 76 positions of the primary structure for the 20 amino acids in segments of four to seven residues defined by secondary structure and selected the folded species for interaction with h-ras by using an in vivo survival-selection assay. The methodology presented allowed for rigorous statistical analysis and comparison of sequence diversity. The ensemble of sequence variants of Raf ras binding domain obtained have recaptured the diversity observed for the ubiquitin-roll topology. A signature sequence for this fold and the implication of this strategy to protein design and structure prediction are discussed.

A strategy for detecting the conservation of folding-nucleus residues in protein superfamilies.

BACKGROUND: Nucleation-growth theory predicts that fast-folding peptide sequences fold to their native structure via structures in a transition-state ensemble that share a small number of native contacts (the folding nucleus). Experimental and theoretical studies of proteins suggest that residues participating in folding nuclei are conserved among homologs. We attempted to determine if this is true in proteins with highly diverged sequences but identical folds (superfamilies). RESULTS: We describe a strategy based on comparisons of residue conservation in natural superfamily sequences with simulated sequences (generated with a Monte-Carlo sequence design strategy) for the same proteins. The basic assumptions of the strategy were that natural sequences will conserve residues needed for folding and stability plus function, the simulated sequences contain no functional conservation, and nucleus residues make native contacts with each other. Based on these assumptions, we identified seven potential nucleus residues in ubiquitin superfamily members. Non-nucleus conserved residues were also identified; these are proposed to be involved in stabilizing native interactions. We found that all superfamily members conserved the same potential nucleus residue positions, except those for which the structural topology is significantly different. CONCLUSIONS: Our results suggest that the conservation of the nucleus of a specific fold can be predicted by comparing designed simulated sequences with natural highly diverged sequences that fold to the same structure. We suggest that such a strategy could be used to help plan protein folding and design experiments, to identify new superfamily members, and to subdivide superfamilies further into classes having a similar folding mechanism.

Visualization of biochemical networks in living cells.

Functional annotation of novel genes can be achieved by detection of interactions of their encoded proteins with known proteins followed by assays to validate that the gene participates in a specific cellular function. We report an experimental strategy that allows for detection of protein interactions and functional assays with a single reporter system. Interactions among biochemical network component proteins are detected and probed with stimulators and inhibitors of the network. In addition, the cellular location of the interacting proteins is determined. We used this strategy to map a signal transduction network that controls initiation of translation in eukaryotes. We analyzed 35 different pairs of full-length proteins and identified 14 interactions, of which five have not been observed previously, suggesting that the organization of the pathway is more ramified and integrated than previously shown. Our results demonstrate the feasibility of using this strategy in efforts of genomewide functional annotation.

A mechanism for rotamase catalysis by the FK506 binding protein (FKBP).

A detailed mechanism for the catalysis of prolyl isomerization by the rotamase enzyme FKBP is proposed on the basis of a model constructed from the known structure of the FK506/FKBP complex. The model substrate is bound as a type VIa proline turn with the ends exposed to permit longer polypeptide chains (e.g., protein loops) to act as substrates. An ab initio potential for the isomerized imide bond is combined with a molecular mechanics representation of the rest of the system to calculate the reaction path. The resulting activation energy for the enzymatic cis-->trans isomerization is equal to about 6 kcal/mol, in good agreement with experiment. The lowering of the barrier relative to the solution value of 19 kcal/mol is found to arise from a combination of desolvation of the imide carbonyl, ground-state destabilization, substrate autocatalysis, and preferential transition-state binding. Minimal rearrangements are required in the enzyme and the substrate along the reaction path. The enzyme residues that participate in catalysis agree with the available mutation data. The type VIa turn model corresponds to a sequence-specific structural motif commonly found on the surface of proteins. It is likely to have a role in the formation of protein complexes with FKBP-like domains that function as foldases or chaperones.

Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays.

Two strategies are described for detecting constitutive or induced protein-protein interactions in intact mammalian cells; these strategies are based on oligomerization domain-assisted complementation of rationally designed fragments of the murine enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3). We describe a dominant clonal-selection assay of stably transfected cells expressing partner proteins FKBP (FK506 binding protein) and FRAP (FKBP-rapamycin binding protein) fused to DHFR fragments and show a rapamycin dose-dependent survival of clones that requires approximately 25 molecules of reconstituted DHFR per cell. A fluorescence assay also is described, based on stoichiometric binding of fluorescein-methotrexate to reconstituted DHFR in vivo. Formation of the FKBP-rapamycin-FRAP complex is detected in stably and transiently transfected cells. Quantitative rapamycin dose-dependence of this complex is shown to be consistent with in vitro binding and distinguishable from a known constitutive interaction of FKBP and FRAP. We also show that this strategy can be applied to study membrane protein receptors, demonstrating dose-dependent activation of the erythropoietin receptor by ligands. The combination of these clonal-selection and fluorescence assays in intact mammalian cells makes possible selection by simple survival, flow cytometry, or both. High-throughput drug screening and quantitative analysis of induction or disruption of protein-protein interactions are also made possible.