An important goal after structural genomics is to build up the structures of higher-order protein-protein complexes from structures of the individual subunits. Often structures of higher order complexes are difficult to obtain by crystallography. We have used an alternative approach in which the structures of the individual catalytic (C) subunit and R I ␣ regulatory (R) subunit of PKA were first subjected to computational docking, and the top 100,000 solutions were subsequently filtered based on amide hydrogen͞ deuterium (H͞ 2 H) exchange interface protection data. The resulting set of filtered solutions forms an ensemble of structures in which, besides the inhibitor peptide binding site, a flat interface between the C-terminal lobe of the C-subunit and the A-and B-helices of R I ␣ is uniquely identified. This holoenzyme structure satisfies all previous experimental data on the complex and allows prediction of new contacts between the two subunits.matrix-assisted laser desorption ionization/time-of-flight ͉ protein-protein interactions ͉ PKA holoenzyme (type I) P rotein kinase A (PKA), a central locus for cAMP-mediated signaling in mammalian cells, exists in an inactive state as a tetrameric holoenzyme composed of two regulatory (R) and two catalytic (C) subunits. Binding of cAMP to the R-subunits leads to dissociation of the holoenzyme and unleashing of the active C-subunit. Like many protein kinases, inhibition of the Csubunit by the R I ␣ regulatory subunit occurs by bipartite binding to the C-subunit. The R-subunit has a pseudosubstrate͞inhibitor sequence near the N terminus that binds in the active site of the C-subunit. Two cAMP-binding domains (A and B) follow the pseudosubstrate͞inhibitor sequence, and it is the A-domain of the R I ␣ isoform that contains the peripheral binding site for inhibition of the C-subunit (1, 2). Although crystal structures are available for the C-subunit in various active and inhibited forms (3), and for the cAMP-bound R I ␣(113-376) (4), no structural information is available for the holoenzyme complex consisting of the R-subunit and the C-subunit.Computational docking is a useful approach to build up the structures of larger protein-protein complexes. The program DOT computes the interaction energies of all possible (billions) translational and rotational solutions by using fast Fourier transform algorithms (5, 6). The interaction between the Csubunit and the R-subunit involves two sites, each of which contributes to the overall binding energy, and DOT did not predict a unique docking interface. To filter the results, we first tried using results from complementary mutagenesis experiments, which revealed a contact between Lys-213 in the Csubunit with Glu-143 in the R-subunit (7). This contact alone, however, was insufficient to specify a unique solution (I.T. and L.T.E., unpublished results).Amide H͞ 2 H exchange, followed by proteolytic digestion and mass spectrometry, is a powerful method to map protein-protein and protein-ligand interactions (8-11). Amide H͞ 2 H exchange c...

Lambda repressor fragment λ(*)(6-85) is one of the fastest folding small protein fragments known to date. We hypothesized that removal of three out of five helices of λ(*)(6-85) would further reduce this protein to its smallest folding core. Molecular dynamics simulations singled out two energetically stable reduced structures consisting of only helices 1 and 4 connected by a short glycine/serine linker, as well as a less stable control. We investigated these three polypeptides and their fragments experimentally by using circular dichroism, fluorescence spectroscopy, and temperature jump relaxation spectroscopy to gain insight into their thermodynamic and kinetic properties. Based on the thermal melts, the order of peptide stability was in correspondence with theoretical predictions. The most stable two-helix bundle, λ(blue1), is a cooperatively folding miniprotein with the same melting temperature and folding rate as the full-length λ(*)(6-85) pseudo wild type and a well-defined computed structure.

We present an evaluation of our results for the first Critical Assessment of PRedicted Interaction (CAPRI). The methods used include the molecular docking program DOT, shape analysis tool FADE, cluster analysis and filtering based on biological data. Good results were obtained for most of the seven CAPRI targets, and for two systems, submissions having the highest number of correctly predicted contacts were produced.

The macromolecular docking problem that must be solved for experimental biologists is prediction of the structures of complexes for which the components are known or reliably modeled in the unbound state, but the structure of the complex is unknown. The current state of the art in macromolecular docking is such that solving this problem usually requires supplementary experimental chemical and/or biological information to evaluate computational predictions. Amide (1)H/(2)H exchange measured by mass spectroscopy is a promising approach for obtaining such information, because it can reveal interfacial regions of each member of the complex and identify regions of conformational flexibility in the structure. In a previous article (Anand et al., Proc Natl Acad Sci USA 2003;100:13264-13269), we used (1)H/(2)H exchange data to predict the structure of a complex between regulatory and catalytic subunits of protein kinase A. Comparison of the prediction with a recent crystal structure determination (Kim et al., Science 2005;307:690-696) showed large conformational change in the regulatory subunit on formation of the complex. Analysis of the prediction, previous CAPRI results, novel data processing methods for the (1)H/(2)H exchange data, and new fragment docking computations give grounds for cautious optimism that this method can be useful even in cases of substantial conformational change.