Intramolecular carbohydrate-aromatic interactions and intermolecular van der Waals interactions enhance the molecular recognition ability of GM1 glycomimetics for cholera toxin

The design and synthesis of two GM1 glycomimetics, 6 and 7, and analysis of their conformation in the free state and when complexed to cholera toxin is described. These compounds, which include an (R)-cyclohexyllactic acid and an (R)-phenyllactic acid fragment, respectively, display significant affinity for cholera toxin. A detailed NMR spectroscopy study of the toxin/glycomimetic complexes, assisted by molecular modeling techniques, has allowed their interactions with the toxin to be explained at the atomic level. It is shown that intramolecular van der Waals and CH-pi carbohydrate-aromatic interactions define the conformational properties of 7, which adopts a three-dimensional structure significantly preorganized for proper interaction with the toxin. The exploitation of this kind of sugar-aromatic interaction, which is very well described in the context of carbohydrate/protein complexes, may open new avenues for the rational design of sugar mimics.     

Accelerating the Association of the Most Stable Protein–Ligand Complex by More than Two Orders of Magnitude

The complex between the bacterial type 1 pilus subunit FimG and the peptide corresponding to the N‐terminal extension (termed donor strand, Ds) of the partner subunit FimF (DsF) shows the strongest reported noncovalent molecular interaction, with a dissociation constant (K_D) of 1.5×10^?20 m . However, the complex only exhibits a slow association rate of 330 m ^?1 s^?1 that limits technical applications, such as its use in affinity purification. Herein, a structure‐based approach was used to design pairs of FimGt (a FimG variant lacking its own N‐terminal extension) and DsF variants with enhanced electrostatic surface complementarity. Association of the best mutant FimGt/DsF pairs was accelerated by more than two orders of magnitude, while the dissociation rates and 3D structures of the improved complexes remained essentially unperturbed. A K_D value of 8.8×10^?22 m was obtained for the best mutant complex, which is the lowest value reported to date for a protein/ligand complex.     

Highly Ordered Self‐Assembly of Native Proteins into 1D, 2D,and 3D Structures Modulated by the Tether Length of Assembly‐Inducing Ligands

In nature, proteins self‐assemble into various structures with different dimensions. To construct these nanostructures in laboratories, normally proteins with different symmetries are selected. However, most of these approaches are engineering‐intensive and highly dependent on the accuracy of the protein design. Herein, we report that a simple native protein LecA assembles into one‐dimensional nanoribbons and nanowires, two‐dimensional nanosheets, and three‐dimensional layered structures controlled mainly by small‐molecule assembly‐inducing ligands RnG (n =1, 2, 3, 4, 5) with varying numbers of ethylene oxide repeating units. To understand the formation mechanism of the different morphologies controlled by the small‐molecule structure, molecular simulations were performed from microscopic and mesoscopic view, which presented a clear relationship between the molecular structure of the ligands and the assembled patterns. These results introduce an easy strategy to control the assembly structure and dimension, which could shed light on controlled protein assembly.     

Isothermal titration calorimetry: application to structure-based drug design

The road to market for drug compounds is a treacherous one, generally involving a huge temporal and financial investment. The role of structure-based drug design or lead optimisation ranges wildly in importance in different pharmaceutical companies. The adoption of these aids to provide routes to high affinity ligands has not received widespread acceptance. This is based on a number of factors, from the perceived failings of such methods, to the belief that rapid screening of compound libraries alone is the most effective way to discover drugs.

The panacea of being able to take a computer generated representation of the structure of a target site of a given biomolecule and rationally design an high affinity inhibiting compound has proved seemingly unreachable for three major reasons: (1) current capabilities in computing; (2) the requirement for atomic resolution structural detail; and (3) determination of how structural features can be related to the thermodynamics of interactions. It is the last of these points that this review seeks to address. In particular the use of isothermal titration calorimetry is discussed in the light of the accumulation of accurate thermodynamic data and examples are given where this has been applied to understand the structural aspects of formation of drug–biomolecular complexes.     

Effects of chain length and N-methylation on a cation-pi interaction in a beta-hairpin peptide

The effects of N-methylation and chain length on a cation-pi interaction have been investigated within the context of a beta-hairpin peptide. Significant enhancement of the interaction and structural stabilization of the hairpin have been observed upon Lys methylation. Thermodynamic analysis indicates an increased entropic driving force for folding upon methylation of Lys residues. Comparison of lysine to analogues ornithine (Orn) and diaminobutyric acid (Dab) indicates that lysine provides the strongest cation-pi interaction and also provides the most stable beta-hairpin due to a combination of side chain-side chain interactions and beta-sheet propensities. These studies have significance for the recognition of methylated lysine in histone proteins.     

Designed Asymmetric Protein Assembly on a Symmetric Scaffold

Cellular signaling is regulated by the assembly of proteins into higher‐order complexes. Bottom‐up creation of synthetic protein assemblies, especially asymmetric complexes, is highly challenging. Presented here is the design and implementation of asymmetric assembly of a ternary protein complex facilitated by Rosetta modeling and thermodynamic analysis. The wild‐type symmetric CT32–CT32 interface of the 14‐3‐3–CT32 complex was targeted, ultimately favoring asymmetric assembly on the 14‐3‐3 scaffold. Biochemical studies, supported by mass‐balance models, allowed characterization of the parameters driving asymmetric assembly. Importantly, our work reveals that both the individual binding affinities and cooperativity between the assembling components are crucial when designing higher‐order protein complexes. Enzyme complementation on the 14‐3‐3 scaffold highlighted that interface engineering of a symmetric ternary complex generates asymmetric protein complexes with new functions.     

Solution-phase combinatorial libraries: modulating cellular signaling by targeting protein-protein or protein-DNA interactions

The high-throughput synthesis and screening of compound libraries hold tremendous promise for drug discovery and powerful methods for both solid-phase and solution-phase library preparation have been introduced. The question of which approach (solution-phase versus solid-phase) is best for the preparation of chemical libraries has been replaced by which approach is most appropriate for a particular target or screen. Herein we highlight distinctions in the two approaches that might serve as useful considerations at the onset of new programs. This is followed by a more personal account of our own focus on solution-phase techniques for the preparation of libraries designed to modulate cellular signaling by targeting protein-protein or protein-DNA interactions. The screening of our libraries against a prototypical set of extracellular and intracellular targets, using a wide range of assay formats, provided the first small-molecule modulators of the protein-protein interactions studied, and a generalized approach for conducting such studies.     

An object-oriented library for computational protein design

Recent advances in computational protein design have established it as a viable technique for the rational generation of stable protein sequences, novel protein folds, and even enzymatic activity. We present a new and object-oriented library of code, written specifically for protein design applications in C(++), called EGAD Library. The modular fashion in which this library is written allows developers to tailor various energy functions and minimizers for a specific purpose. It also allows for the generation of novel protein design applications with a minimal amount of code investment. It is our hope that this will permit labs that have not considered protein design to apply it to their own systems, thereby increasing its potential as a tool in biology. We also present various uses of EGAD Library: in the development of Interaction Viewer, a PyMOL plug-in for viewing interactions between protein residues; in the repacking of protein cores; and in the prediction of protein-protein complex stabilities.     

Conformationally constrained sequence designs to bias monomer-dimer equilibriums in TASP systems

We have designed template-assembled synthetic proteins (TASPs) with the intent of controlling their oligomeric state by stabilizing specific helical tertiary structures via histidine metal ion chelation or disulfide incorporation. In solution, cavitein Q4 was previously determined to interconvert between a four-helix bundle monomer and an eight-helix bundle dimer. In this paper, we show that judicious mutation of cavitein Q4 can stabilize either the monomeric parallel four-helix bundle or the dimeric antiparallel eight-helix bundle structure. Cavitein Q4-E3H, designed to be dimeric, is indeed biased toward dimerization as a result of incorporation of histidines. Moreover, the addition of nickel was found to further increase the association constant of dimerization. Similarly, a cavitein designed to stabilize the monomeric structure via histidine metal ion chelation (Q4-H) was found to favor a monomer in solution upon addition of nickel. Lastly, a cavitein intended to stabilize a monomeric structure via disulfide incorporation (Q4-C2) is reported. Surprisingly, this disulfide cavitein yielded two products upon oxidation suggesting disulfide formation both above the cavitand template and below may be possible. Nevertheless, the two disulfide caviteins were shown to exist as monomers as per their design.     

A Designed Conformational Shift To Control Protein Binding Specificity

In a conformational selection scenario, manipulating the populations of binding‐competent states should be expected to affect protein binding. We demonstrate how in silico designed point mutations within the core of ubiquitin, remote from the binding interface, change the binding specificity by shifting the conformational equilibrium of the ground‐state ensemble between open and closed substates that have a similar population in the wild‐type protein. Binding affinities determined by NMR titration experiments agree with the predictions, thereby showing that, indeed, a shift in the conformational equilibrium enables us to alter ubiquitin’s binding specificity and hence its function. Thus, we present a novel route towards designing specific binding by a conformational shift through exploiting the fact that conformational selection depends on the concentration of binding‐competent substates.     

Cross‐Linking/Mass Spectrometry for Studying Protein Structures and Protein–Protein Interactions: Where Are We Now and Where Should We Go from Here?

Structural mass spectrometry (MS) is gaining increasing importance for deriving valuable three‐dimensional structural information on proteins and protein complexes, and it complements existing techniques, such as NMR spectroscopy and X‐ray crystallography. Structural MS unites different MS‐based techniques, such as hydrogen/deuterium exchange, native MS, ion‐mobility MS, protein footprinting, and chemical cross‐linking/MS, and it allows fundamental questions in structural biology to be addressed. In this Minireview, I will focus on the cross‐linking/MS strategy. This method not only delivers tertiary structural information on proteins, but is also increasingly being used to decipher protein interaction networks, both in vitro and in vivo. Cross‐linking/MS is currently one of the most promising MS‐based approaches to derive structural information on very large and transient protein assemblies and intrinsically disordered proteins.     

Do Intrinsically Disordered Proteins Possess High Specificity in Protein–Protein Interactions?

Specific protein–protein interactions are critical to cellular function. Structural flexibility and disorder‐to‐order transitions upon binding enable intrinsically disordered proteins (IDPs) to overcome steric restrictions and form complementary binding interfaces, and thus, IDPs are widely considered to have high specificity and low affinity for molecular recognition. However, flexibility may also enable IDPs to form complementary binding interfaces with misbinding partners, resulting in a great number of nonspecific interactions. Consequently, it is questionable whether IDPs really possess high specificity. In this work, we investigated this question from a thermodynamic viewpoint. We collected mutant thermodynamic data for 35 ordered protein complexes and 43 disordered protein complexes. We found that the enthalpy–entropy compensation for disordered protein complexes was more complete than that for ordered protein complexes. We further simulated the binding processes of ordered and disordered protein complexes under mutations. Simulation data confirmed the observation of experimental data analyses and further revealed that disordered protein complexes possessed smaller changes in binding free energy than ordered protein complexes under the same mutation perturbations. Therefore, interactions of IDPs are more malleable than those of ordered proteins due to their structural flexibility in the complex. Our results provide new clues for exploring the relationship between protein flexibility, adaptability, and specificity.     

Phosphopeptide-dependent labeling of 14-3-3 ζ proteins by fusicoccin-based fluorescent probes

Fluorescent combination: Cell-penetrating probes derived from the diterpene fusicoccin can form ternary complexes with 14-3-3 proteins and phosphopeptide ligands, whereupon the probes site-specifically attach a fluorescent tag onto the surface of the 14-3-3 proteins.