Lynn F. Ten Eyck

CAPRI: A Critical Assessment of PRedicted Interactions

CAPRI is a communitywide experiment to assess the capacity of protein‐docking methods to predict protein–protein interactions. Nineteen groups participated in rounds 1 and 2 of CAPRI and submitted blind structure predictions for seven protein–protein complexes based on the known structure of the component proteins. The predictions were compared to the unpublished X‐ray structures of the complexes. We describe here the motivations for launching CAPRI, the rules that we applied to select targets and run the experiment, and some conclusions that can already be drawn. The results stress the need for new scoring functions and for methods handling the conformation changes that were observed in some of the target systems. CAPRI has already been a powerful drive for the community of computational biologists who development docking algorithms. We hope that this issue of Proteins will also be of interest to the community of structural biologists, which we call upon to provide new targets for future rounds of CAPRI, and to all molecular biologists who view protein–protein recognition as an essential process. Proteins 2003;52:2–9.

Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism

The surface comparison of different serine–threonine and tyrosine kinases reveals a set of 30 residues whose spatial positions are highly conserved. The comparison between active and inactive conformations identified the residues whose positions are the most sensitive to activation. Based on these results, we propose a model of protein kinase activation. This model explains how the presence of a phosphate group in the activation loop determines the position of the catalytically important aspartate in the Asp-Phe-Gly motif. According to the model, the most important feature of the activation is a “spine” formation that is dynamically assembled in all active kinases. The spine is comprised of four hydrophobic residues that we detected in a set of 23 eukaryotic and prokaryotic kinases. It spans the molecule and plays a coordinating role in activated kinases. The spine is disordered in the inactive kinases and can explain how stabilization of the whole molecule is achieved upon phosphorylation.

A helix scaffold for the assembly of active protein kinases

Structures of set of serine-threonine and tyrosine kinases were investigated by the recently developed bioinformatics tool Local Spatial Patterns (LSP) alignment. We report a set of conserved motifs comprised mostly of hydrophobic residues. These residues are scattered throughout the protein sequence and thus were not previously detected by traditional methods. These motifs traverse the conserved protein kinase core and play integrating and regulatory roles. They are anchored to the F-helix, which acts as an organizing “hub” providing precise positioning of the key catalytic and regulatory elements. Consideration of these discovered structures helps to explain previously inexplicable results.

Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis.

Proteins that undergo cooperative unfolding events display EX1 kinetic signatures in hydrogen exchange mass spectra. The hallmark bimodal isotope pattern observed for EX1 kinetics is distinct from the binomial isotope pattern for uncorrelated exchange (EX2), the normal exchange regime for folded proteins. Detection and characterization of EX1 kinetics is simple when the cooperative unit is large enough that the isotopic envelopes in the bimodal pattern are resolved in the m/z scale but become complicated in cases where the unit is small or there is a mixture of EX1 and EX2 kinetics. Here we describe a data interpretation method involving peak width analysis that makes characterization of EX1 kinetics simple and rapid. The theoretical basis for EX1 and EX2 isotopic signatures and the effects each have on peak width are described. Modeling of EX2 widening and analysis of empirical data for proteins and peptides containing purely EX2 kinetics showed that the amount of widening attributable to stochastic forward- and back exchange in a typical experiment is small and can be quantified. Proteins and peptides with both obvious and less obvious EX1 kinetics were analyzed with the peak width method. Such analyses provide the half-life for the cooperative unfolding event and the relative number of residues involved. Automated analysis of peak width was performed with custom Excel macros and the DEX software package. Peak width analysis is robust, capable of automation, and provides quick interpretation of the key information contained in EX1 kinetic events.

Identification of the protein kinase A regulatory RIα-catalytic subunit interface by amide H/2H exchange and protein docking

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 RIα regulatory (R) subunit of PKA were first subjected to computational docking, and the top 100,000 solutions were subsequently filtered based on amide hydrogen/deuterium (H/2H) 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 RIα is uniquely identified. This holoenzyme structure satisfies all previous experimental data on the complex and allows prediction of new contacts between the two subunits.

Automated extraction of backbone deuteration levels from amide H/2H mass spectrometry experiments

A Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high‐resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side‐chain hydrogens (present in MALDI‐TOF H/2H data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side‐chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances.

Surveying Molecular Interactions with DOT

The purpose of the molecular interaction program DOT (Daughter of Turnip) is rapid computation of the electrostatic potential energy between two proteins or other charged molecules. DOT exhaustively tests all six degrees of freedom, rotational and translational, and produces a grid of approximate interaction energies and orientations. It is able to do this because the problem is cast as the convolution of the potential field of the first molecule and any rotated charge distribution of the second. The algorithm lends itself to both parallelization and vectorization, permitting huge increases in computational speed over other methods for obtaining the same information. For example, a complete mapping of interactions between plastocyanin and cytochrome c was done in eight minutes using 256 nodes of an Intel Paragon. DOT is expected to be particularly useful as a rapid screen to find configurations for more detailed study using exact energy models.

Rapid atomic density methods for molecular shape characterization.

Two methods for rapid characterization of molecular shape are presented. Both techniques are based on the density of atoms near the molecular surface. The Fast Atomic Density Evaluation (FADE) algorithm uses fast Fourier transforms to quickly estimate densities. The Pairwise Atomic Density Reverse Engineering (PADRE) method derives modified density measures from the relationship between atomic density and total potentials. While many shape-characterization techniques define shape relative to a surface, the descriptors returned by FADE and PADRE can measure local geometry from points within the three-dimensional space surrounding a molecule. The methods can be used to find crevices and protrusions near the surface of a molecule and to test shape complementarity at the interface between docking molecules.

A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains.

Cyclic nucleotides (cAMP and cGMP) regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB) domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels) using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1) the phosphate binding cassette (PBC), which binds the cAMP ribose-phosphate, 2) the “hinge,” a flexible helix, which contacts the PBC, 3) the β2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4) a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif). The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the β2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP) represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.

Catalytic Subunit of Cyclic AMP-Dependent Protein Kinase: Structure and Dynamics of the Active Site Cleft

The catalytic subunit of cyclic AMP-dependent protein kinase serves as a structural template for the entire family of Ser, Thr, and Tyr specific protein kinases. We review here the dynamics of the active catalytic subunit. These dynamics correlate with an opening and closing of the active site cleft, and are considered to be a requirement for catalysis. The motions, described by a set of several crystal structures, reveal a very fluid active site cleft. This active site cleft with its dynamic opening and closing is a prime target for protein kinase inhibitors.