PDZ domains have long been thought to cluster into discrete functional classes defined by their peptide-binding preferences. We used protein microarrays and quantitative fluorescence polarization to characterize the binding selectivity of 157 mouse PDZ domains with respect to 217 genome-encoded peptides. We then trained a multidomain selectivity model to predict PDZ domain-peptide interactions across the mouse proteome with an accuracy that exceeds many large-scale, experimental investigations of protein-protein interactions. Contrary to the current paradigm, PDZ domains do not fall into discrete classes; instead, they are evenly distributed throughout selectivity space, which suggests that they have been optimized across the proteome to minimize cross-reactivity. We predict that focusing on families of interaction domains, which facilitates the integration of experimentation and modeling, will play an increasingly important role in future investigations of protein function.

The signaling network downstream of the ErbB family of receptors has been extensively targeted by cancer therapeutics; however, understanding the relative importance of the different components of the ErbB network is nontrivial. To explore the optimal way to therapeutically inhibit combinatorial, ligand-induced activation of the ErbB-phosphatidylinositol 3-kinase (PI3K) axis, we built a computational model of the ErbB signaling network that describes the most effective ErbB ligands, as well as known and previously unidentified ErbB inhibitors. Sensitivity analysis identified ErbB3 as the key node in response to ligands that can bind either ErbB3 or EGFR (epidermal growth factor receptor). We describe MM-121, a human monoclonal antibody that halts the growth of tumor xenografts in mice and, consistent with model-simulated inhibitor data, potently inhibits ErbB3 phosphorylation in a manner distinct from that of other ErbB-targeted therapies. MM-121, a previously unidentified anticancer therapeutic designed using a systems approach, promises to benefit patients with combinatorial, ligand-induced activation of the ErbB signaling network that are not effectively treated by current therapies targeting overexpressed or mutated oncogenes.

Early protein synthesis is thought to have involved a reduced amino acid alphabet. What is the minimum number of amino acids that would have been needed to encode complex protein folds similar to those found in nature today? Here we show that a small beta-sheet protein, the SH3 domain, can be largely encoded by a five letter amino acid alphabet but not by a three letter alphabet. Furthermore, despite the dramatic changes in sequence, the folding rates of the reduced alphabet proteins are very close to that of the naturally occurring SH3 domain. This finding suggests that despite the vast size of the search space, the rapid folding of biological sequences to their native states is not the result of extensive evolutionary optimization. Instead, the results support the idea that the interactions which stabilize the native state induce a funnel shape to the free energy landscape sufficient to guide the folding polypeptide chain to the proper structure.

Although human epidermal growth factor receptor 2 (HER2) overexpression is implicated in tumor progression for a variety of cancer types, how it dysregulates signaling networks governing cell behavioral functions is poorly understood. To address this problem, we use quantitative mass spectrometry to analyze dynamic effects of HER2 overexpression on phosphotyrosine signaling in human mammary epithelial cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). Data generated from this analysis reveal that EGF stimulation of HER2-overexpressing cells activates multiple signaling pathways to stimulate migration, whereas HRG stimulation of these cells results in amplification of a specific subset of the migration signaling network. Self-organizing map analysis of the phosphoproteomic data set permitted elucidation of network modules differentially regulated in HER2-overexpressing cells in comparison with parental cells for EGF and HRG treatment. Partial least-squares regression analysis of the same data set identified quantitative combinations of signals within the networks that strongly correlate with cell proliferation and migration measured under the same battery of conditions. Combining these modeling approaches enabled association of epidermal growth factor receptor family dimerization to activation of specific phosphorylation sites, which appear to most critically regulate proliferation and/or migration.

Experimental and theoretical studies on the folding of small proteins such as the chymotrypsin inhibitor 2 (CI-2) and the P22 Arc repressor suggest that the folding transition state is an expanded version of the native state with most interactions partially formed. Here we report that this picture does not hold generally: a hydrogen bond network involving two beta-turns and an adjacent hydrophobic cluster appear to be formed in the folding transition state of the src SH3 domain, while the remainder of the polypeptide chain is largely unstructured. Comparison with data on other small proteins suggests that this structural polarization is a consequence of the topology of the SH3 domain fold. The non-uniform distribution of structure in the folding transition state provides a challenging test for computational models of the folding process.

No abstract

The thermodynamics and kinetics of folding of the chicken src SH3 domain were characterized using equilibrium and stopped-flow fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) hydrogen exchange experiments. As found for other SH3 domains, guanidinium chloride (GdmCl) denaturation melts followed by both fluorescence and circular dichroism were nearly superimposable, indicating the concerted formation of secondary and tertiary structure. Kinetic studies confirmed the two-state character of the folding reaction. Except for a very slow refolding phase due to proline isomerization, both folding and unfolding traces fit well to single exponentials over a wide range of GdmCl concentrations, and no burst phase in amplitude was observed during the dead time of the stopped-flow instrument. The entropy, enthalpy, and heat capacity changes upon unfolding were determined by global fitting of temperature melts at varying GdmCl concentrations (0.4-3.7 M). Estimates of the free energy of unfolding, DeltaGUH2O, from guanidine denaturation, thermal denaturation, and kinetic experiments were in good agreement. To complement these data on the global characteristics of src SH3 folding, individual hydrogen-deuterium (HD) exchange rates were measured for approximately half of the backbone amides in 0 and 0.7 M GdmCl. The calculated free energies of the opening reaction leading to exchange (DeltaGHD) indicated that unfolding is highly cooperative--slowly exchanging protons were distributed throughout the core of the protein. The slowly exchanging protons exhibited DeltaGHD values higher than the global DeltaGUH2O by approximately 1 kcal/mol, suggesting that the denatured state might be somewhat compact under native conditions. Comparison of the src SH3 with homologous SH3 domains as well as with other small well-characterized beta-sheet proteins provides insights into the determinants of folding kinetics and protein stability.

One of the outstanding questions in protein folding concerns the degree of heterogeneity in the folding transition state ensemble: does a protein fold via a large multitude of diverse ''pathways,'' or are the elements of native structure assembled in a well defined order? Herein, we build on previous point mutagenesis studies of the src SH3 by directly investigating the association of structural elements and the loss of backbone conformational entropy during folding. Double-mutant analysis of polar residues in the distal ␤-hairpin and the diverging turn indicates that the hydrogen bond network between these elements is largely formed in the folding transition state. A 10-glycine insertion in the n-src loop (which connects the distal hairpin and the diverging turn) and a disulfide crosslink at the base of the distal ␤-hairpin exclusively affect the folding rate, showing that these structural elements are nearly as ordered in the folding transition state as in the native state. In contrast, crosslinking the base of the RT loop or the N and C termini dramatically slows down the unfolding rate, suggesting that dissociation of the termini and opening of the RT loop precede the rate-limiting step in unfolding. Taken together, these results suggest that essentially all conformations in the folding transition state ensemble have the central three-stranded ␤-sheet formed, indicating that, for the src homology 3 domain, there is a discrete order to structure assembly during folding.