The prediction of binding sites and the understanding of interfaces associated with protein complexation remains an open problem in molecular biophysics. This work shows that a crucial factor in predicting and rationalizing protein-protein interfaces can be inferred by assessing the extent of intramolecular desolvation of backbone hydrogen bonds in monomeric structures. Our statistical analysis of native structures shows that, in the majority of soluble proteins, most backbone hydrogen bonds are thoroughly wrapped intramolecularly by nonpolar groups except for a few ones. These latter underwrapped hydrogen bonds may be dramatically stabilized by removal of water. This fact implies that packing defects are ''sticky'' in a way that decisively contributes to determining the binding sites for proteins, as an examination of numerous complexes demonstrates.hydrophobic effect ͉ protein structure ͉ protein-ligand association ͉ binding site A theory of hydrophobic interactions (1) based on a statistical mechanical treatment of liquid H 2 O (2) and aqueous solutions of hydrocarbons (3) demonstrated how the removal of water from the neighborhood of nonpolar groups enhanced their interaction free energy in aqueous solution (4). Such dehydration-based hydrophobic interactions enhance the role of nearby intramolecular hydrogen bonds in stabilizing protein conformations (5-9) and facilitating the folding process Fig. 1). It therefore is necessary to provide a systematic description of the nonpolar environments of hydrogen bonds, their variations among native structures, and their evolution during conformational changes. This is needed, for example, to assess the role of water removal in protein-ligand associations (13, 14), molecular disease, and aggregation (15,16). To address such problems, we define a hydrogen-bond dehydration domain and count the number of nonpolar groups within. We show that a field must be introduced to account for spots on the protein surface where water exclusion resulting from intergroup interaction plays a key role in strengthening nearby hydrogen bonds. Such hot spots enhance the contribution of hydrophobic interactions and contribute to defining binding sites, nucleating sites for aggregation, and protein reactivity in general.The dehydration of backbone hydrogen bonds by nearby nonpolar groups makes it thermodynamically unfavorable to expose the backbone amide and carbonyl groups (Fig. 1). Similarly, as shown in Fig. 1, nearby nonpolar groups enhance the dehydration of the nonpolar parts of polar side chains as well as restricting the rotational freedom of the polar side chain, thereby increasing the stability of side-chain hydrogen bonds. Thus, the stabilization of secondary structure generally requires a higher-order organization of the chain to dehydrate the hydrogen bonds (10-12), shielding them from water attack. In view of this, we expect that most native structures of soluble proteins in their monomeric form would have most of their hydrogen bonds thoroughly dehydrated to warrant their ov...

The boundaries between prokaryotes, unicellular eukaryotes, and multicellular eukaryotes are accompanied by orders-of-magnitude reductions in effective population size, with concurrent amplifications of the effects of random genetic drift and mutation1. The resultant decline in the efficiency of selection appears to be sufficient to influence a wide range of attributes at the genomic level in a nonadaptive manner2. A key remaining question concerns the extent to which variation in the power of random genetic drift is capable of influencing phylogenetic diversity at the subcellular and cellular levels2–4. Should this be the case, population size would have to be considered as a potential determinant of the mechanistic pathways underlying long-term phenotypic evolution. Here we demonstrate a phylogenetically broad inverse relationship between the power of drift and the structural integrity of protein subunits. This leads to the hypothesis that the accumulation of mildly deleterious mutations in populations of small size induces secondary selection for protein-protein interactions that stabilize key gene functions. By this means, the complex protein architectures and interactions essential to the genesis of phenotypic diversity may initially emerge by nonadaptive mechanisms.

Thermosensors are ubiquitous integral membrane proteins found in all kinds of life. They are involved in many physiological roles, including membrane remodeling, chemotaxis, touch, and pain [1-3], but, the mechanism by which their transmembrane (TM) domains transmit temperature signals is largely unknown. The histidine kinase DesK from Bacillus subtilis is the paradigmatic example of a membrane-bound thermosensor suited to remodel membrane fluidity when the temperature drops below approximately 30°C [1, 4] providing, thus, a tractable system for investigating the mechanism of TM-mediated input-output control of thermal adaptation. Here we show that the multimembrane-spanning domain from DesK can be simplified into a chimerical single-membrane-spanning minimal sensor (MS) that fully retains, in vivo and in vitro, the sensing properties of the parental system. The MS N terminus contains three hydrophilic amino acids near the lipid-water interface creating an instability hot spot. Mutational analysis of this boundary-sensitive beacon revealed that membrane thickness controls the signaling state of the sensor by dictating the hydration level of the metastable hydrophilic spot. Guided by these results we biochemically demonstrated that the MS signal transmission activity is sensitive to bilayer thickness. Membrane thickness could be a general cue for sensing temperature in many organisms.

We introduce a semiempirical approach to ab initio prediction of expeditious pathways and native backbone geometries of proteins folding under in vitro renaturation conditions. The algorithm incorporates a discretized codification of local steric hindrances which constrain the movements of the peptide backbone. Thus, torsional motion is shown to be conditioned by the hopping from one basin of attraction (R-basin) to another in the Ramachandran map or local potential energy surface associated with each residue. Rather than simulating detailed dynamics, we simulate the time evolution of such torsional constraints. The semiempirical potential needed to obtain geometric realizations of such “modulo R-basin” topologies is rescaled with each iteration of the simulation in order to incorporate the role of conformation-dependent local environments. Thus, the extent of local desolvation within which a specific interaction occurs is computed for each iteration using an effective local “solvophobic field” determined by two-body interactions emerging from the previous iteration. The predictive power of the algorithm is established by (a) computing ab initio folding pathways for mammalian ubiquitin that yield a stable structural pattern reproducing all of its native features in spite of some adverse local propensities associated with those features when taken in isolation; (b) determining the nucleating event that triggers the hydrophobic collapse of the chain; and (c) comparing coarse predictions of stable folds of moderate size proteins (N∼100) with structures from the Protein Data Bank.

Targeting kinases is central to drug-based cancer therapy but remains challenging because the drugs often lack specificity, which may cause toxic side effects. Modulating side effects is difficult because kinases are evolutionarily and hence structurally related. The lack of specificity of the anticancer drug imatinib enables it to be used to treat chronic myeloid leukemia, where its target is the Bcr-Abl kinase, as well as a proportion of gastrointestinal stromal tumors (GISTs), where its target is the C-Kit kinase. However, imatinib also has cardiotoxic effects traceable to its impact on the C-Abl kinase. Motivated by this finding, we made a modification to imatinib that hampers Bcr-Abl inhibition; refocuses the impact on the C-Kit kinase; and promotes inhibition of an additional target, JNK, a change that is required to reinforce prevention of cardiotoxicity. We established the molecular blueprint for target discrimination in vitro using spectrophotometric and colorimetric assays and through a phage-displayed kinase screening library. We demonstrated controlled inhibitory impact on C-Kit kinase in human cell lines and established the therapeutic impact of the engineered compound in a novel GIST mouse model, revealing a marked reduction of cardiotoxicity. These findings identify the reengineered imatinib as an agent to treat GISTs with curbed side effects and reveal a bottom-up approach to control drug specificity.

We introduce a quantifiable structural motif, called dehydron, that is shown to be central to protein-protein interactions. A dehydron is a defectively packed backbone hydrogen bond suggesting preformed monomeric structure whose Coulomb energy is highly sensitive to binding-induced water exclusion. Such preformed hydrogen bonds are effectively adhesive, since water removal from their vicinity contributes to their stability. At the structural level, a significant correlation is established between dehydrons and sites for protein complexation, with the HIV-1 capsid protein P24 complexed with antibody light-chain FAB25.3 providing the most dramatic correlation. Furthermore, the number of dehydrons in homologous similar-fold proteins from different species is shown to be a signature of proteomic complexity. The techniques are then applied to higher levels of organization: The formation of the capsid and its organization in picornaviruses correlates strongly with the distribution of dehydrons on the rim of the virus unit. Furthermore, antibody contacts and crystal contacts may be assigned to dehydrons still prevalent after the capsid has been assembled. The implications of the dehydron as an encoded signal in proteomics, bioinformatics, and inhibitor drug design are emphasized.

The architecture of present-day protein interaction networks depends on how protein associations evolved. Here, we explore how and why evolution-related mutations influence protein structure to promote protein associations, and thereby network development. We specifically address two questions: (i) How can protein folds remain conserved while proteins accommodate new binding partnerships as genes duplicate? (ii) What is the structural͞molec-ular basis for hub proteins being the most likely to acquire new connections? The answers stem from the examination of the structure wrapping, or protection from water attack. Wrapping is shown to be a crucial consideration in the exploration and evolution of proteomic interactivity.

We show that water constrained by vicinal hydrophobes undergoes a librational dynamics that lowers the dielectric susceptibility and induces a "redshift" of the relaxation frequency in the hydration shell. The results shed light on the way proteins enhance their intramolecular interactions as they fold or associate.