Zygmunt Stanislaw Derewenda

Tryptophan is the most prominent amino acid found in proteins, with multiple functional roles. Its side chain is made up of the hydrophobic indole moiety, with two groups that act as donors in hydrogen bonds: the N ^ɛ —H group, which is a potent donor in canonical hydrogen bonds, and a polarized C ^δ1 —H group, which is capable of forming weaker, noncanonical hydrogen bonds. Due to adjacent electron-withdrawing moieties, C—H...O hydrogen bonds are ubiquitous in macromolecules, albeit contingent on the polarization of the donor C—H group. Consequently, C ^α —H groups (adjacent to the carbonyl and amino groups of flanking peptide bonds), as well as the C ^ɛ1 —H and C ^δ2 —H groups of histidines (adjacent to imidazole N atoms), are known to serve as donors in hydrogen bonds, for example stabilizing parallel and antiparallel β-sheets. However, the nature and the functional role of interactions involving the C ^δ1 —H group of the indole ring of tryptophan are not well characterized. Here, data mining of high-resolution ( r ≤ 1.5 Å) crystal structures from the Protein Data Bank was performed and ubiquitous close contacts between the C ^δ1 —H groups of tryptophan and a range of electronegative acceptors were identified, specifically main-chain carbonyl O atoms immediately upstream and downstream in the polypeptide chain. The stereochemical analysis shows that most of the interactions bear all of the hallmarks of proper hydrogen bonds. At the same time, their cohesive nature is confirmed by quantum-chemical calculations, which reveal interaction energies of 1.5–3.0 kcal mol ⁻¹ , depending on the specific stereochemistry.

Hydrogen bonds constitute a unique type of non-covalent interaction, with a critical role in biology. Until fairly recently, the canonical view held that these bonds occur between electronegative atoms, typically O and N, and that they are mostly electrostatic in nature. However, it is now understood that polarized C-H groups may also act as hydrogen bond donors in many systems, including biological macromolecules. First recognized from physical chemistry studies, C-H…X bonds were visualized with X-ray crystallography sixty years ago, although their true significance has only been recognized in the last few decades. This review traces the origins of the field and describes the occurrence and significance of the most important C-H…O bonds in proteins and nucleic acids.

Background: Phosphorylation of smooth muscle (SM) myosin regulatory light chain (RLC ₂₀ ) is a critical switch leading to contraction or cell migration. The canonical view held that the only kinase catalyzing this reaction is the short isoform of myosin light chain kinase (MLCK1). Auxiliary kinases may be involved and play a vital role in blood pressure homeostasis. We have previously reported that p90 ribosomal S6 kinase (RSK2) functions as such a kinase, in parallel with the classical MLCK1, contributing ∼25% of the maximal myogenic force in resistance arteries and regulating blood pressure. Here, we take advantage of a MLCK1 null mouse to further test our hypothesis that RSK2 can function as an MLCK, playing a significant physiological role in SM contractility. Methods: Fetal (E14.5-18.5) SM tissues were used as embryos die at birth. We investigated the necessity of MLCK for contractility, cell migration and fetal development and determined the ability of RSK2 kinase to compensate for the lack of MLCK and characterized it’s signaling pathway in SM. Results: Agonists induced contraction and RLC ₂₀ phosphorylation in mylk1 ^-/- SM, that was inhibited by RSK2 inhibitors. Embryos developed and cells migrated in the absence of MLCK. The pCa-tension relationships in WT vs mylk1 ^-/- muscles demonstrated a Ca ²⁺ -dependency due to the Ca ²⁺ -dependent tyrosine kinase Pyk2, known to activate PDK1 that phosphorylates and fully activates RSK2. The magnitude of contractile responses was similar upon addition of GTPγS to activate the RhoA/ROCK pathway. The Ca ²⁺ -independent component was through activation of Erk1/2/PDK1/RSK2 leading to direct phosphorylation of RLC ₂₀ , to increase contraction. RSK2, PDK1, Erk1/2 and MLCK formed a signaling complex on the actin filament, optimally positioning them for interaction with adjacent myosin heads. Conclusions: RSK2 signaling constitutes a new third signaling pathway, in addition to the established Ca ²⁺ /CAM/MLCK and RhoA/ROCK pathways to regulate SM contractility and cell migration.

The bent structure of the water molecule, and its hydrogen-bonding properties, arguably rank among the most impactful discoveries in the history of chemistry. Although the fact that the H—O—H angle must deviate from linearity was inferred early in the 20th century, notably from the existence of the electric dipole moment, it was not clear what that angle should be and why. One hundred years ago, a young PhD student at the University of California, Berkeley, Eustace J. Cuy, rationalized the V-shape structure of a water molecule using the Lewis theory of a chemical bond, i.e. a shared electron pair, and its tetrahedral stereochemistry. He was inspired, in part, by the proposal of a weak (hydrogen) bond in water by two colleagues at Berkeley, Wendell Latimer and Worth Rodebush, who published their classic paper a year earlier. Cuy went on to suggest that other molecules, notably H₂S and NH₃, have similar structures, and presciently predicted that this architecture has broader consequences for the structure of water as a liquid. This short, but brilliant paper has been completely forgotten, perhaps due to the tragic death of the author at the age of 28; the hydrogen-bond study is also rarely recognized. One of the most impactful publications on the structure of liquid water, a classic treatise published in 1933 by John Bernal and Ralph Fowler, does not mention either of the two pioneering papers. In this essay, the background for the two discoveries is described, including the brief history of Lewis's research on the nature of the chemical bond, and the history of the discovery of the hydrogen bond, which inspired Cuy to look at the structure of the water molecule. This is – to the best of the author's knowledge – the first biographical sketch of Eustace J. Cuy.

C─H⋯O hydrogen bonds constitute a unique class of cohesive interactions. Their properties are similar to those of canonical H‐bonds, although their energy is significantly lower, typically in the 0.5–2.5 kcal/mol range. Polarised C─H groups, such as those adjacent to electronegative groups, or within aromatic moieties, are particularly strong donors. C─H⋯O bonds are ubiquitous in nucleic acids and in proteins, notably stabilizing the β‐sheet secondary structure. They have also been observed in numerous protein‐ligand interactions. Here, we analysed crystal structures, deposited in the Protein Data Bank, of complexes of FDA‐approved protein kinase inhibitors with cognate kinases, to assess the possible role of C─H^inhibitor⋯O^protein hydrogen bonds. The conserved hinge motif of protein kinases with two solvent‐exposed carbonyl groups and one exposed backbone amide, is well known to be involved in canonical H‐bonding with inhibitors. We now find that in virtually all complexes where the inhibitor interacts with the hinge backbone, at least one of the hinge carbonyl groups accepts an H‐bond from a C─H inhibitor group, which is either aromatic or adjacent to an electronegative group. These observations are important for design of hinge‐binding scaffolds of novel kinase inhibitors for therapeutic use.

The vast majority of platforms for the detection of viral or bacterial antigens rely on immunoassays, typically ELISA or sandwich ELISA, that are contingent on the availability of suitable monoclonal antibodies (mAbs). This is a major bottleneck, since the generation and production of mAbs is time-consuming and expensive. Synthetic antibody fragments (sFabs) generated by phage-display selection offer an alternative with many advantages over Fabs obtained from natural antibodies using hybridoma technology. Unlike mAbs, sFabs are generated using phage display, allowing selection for binding to specific strains or for pan-specificity, for identification of structural epitopes or unique protein conformations and even for complexes. Further, they can easily be produced in Escherichia coli in large quantities and engineered for purposes of detection technologies and other applications. Here, the use of phage-display selection to generate a pan-specific Fab (MJ20), based on a Herceptin Fab scaffold, with the ability to bind selectively and with high affinity to the C-terminal domains of the nucleoproteins (NPs) from all five known strains of the Ebola virus is reported. The high-resolution crystal structure of the complex of MJ20 with the antigen from the Bundibugyo strain of the Ebola virus reveals the basis for pan-specificity and illustrates how the phage-display technology can be used to manufacture suitable Fabs for use in diagnostic or therapeutic applications.

Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

Crystallization of macromolecules has long been perceived as a stochastic process, which cannot be predicted or controlled. This is consistent with another popular notion that the interactions of molecules within the crystal, i.e., crystal contacts, are essentially random and devoid of specific physicochemical features. In contrast, functionally relevant surfaces, such as oligomerization interfaces and specific protein–protein interaction sites, are under evolutionary pressures so their amino acid composition, structure, and topology are distinct. However, current theoretical and experimental studies are significantly changing our understanding of the nature of crystallization. The increasingly popular “sticky patch” model, derived from soft matter physics, describes crystallization as a process driven by interactions between select, specific surface patches, with properties thermodynamically favorable for cohesive interactions. Independent support for this model comes from various sources including structural studies and bioinformatics. Proteins that are recalcitrant to crystallization can be modified for enhanced crystallizability through chemical or mutational modification of their surface to effectively engineer “sticky patches” which would drive crystallization. Here, we discuss the current state of knowledge of the relationship between the microscopic properties of the target macromolecule and its crystallizability, focusing on the “sticky patch” model. We discuss state-of-the-art in silico methods that evaluate the propensity of a given target protein to form crystals based on these relationships, with the objective to design variants with modified molecular surface properties and enhanced crystallization propensity. We illustrate this discussion with specific cases where these approaches allowed to generate crystals suitable for structural analysis.

Ribosomal S6 kinases (RSK) play important roles in cell signaling through the mitogen-activated protein kinase (MAPK) pathway. Each of the four RSK isoforms (RSK1-4) is a single polypeptide chain containing two kinase domains connected by a linker sequence with regulatory phosphorylation sites. Here, we demonstrate that full-length RSK2—which is implicated in several types of cancer, and which is linked to the genetic Coffin-Lowry syndrome—can be overexpressed with high yields in Escherichia coli as a fusion with maltose binding protein (MBP), and can be purified to homogeneity after proteolytic removal of MBP by affinity and size-exclusion chromatography. The purified protein can be fully activated in vitro by phosphorylation with protein kinases ERK2 and PDK1. Compared to full-length RSK2 purified from insect host cells, the bacterially expressed and phosphorylated murine RSK2 shows the same levels of catalytic activity after phosphorylation, and sensitivity to inhibition by RSK-specific inhibitor SL0101. Interestingly, we detect low levels of phosphorylation in the nascent RSK2 on Ser386, owing to autocatalysis by the C-terminal domain, independent of ERK. This observation has implications for in vivo signaling, as it suggests that full activation of RSK2 by PDK1 alone is possible, circumventing at least in some cases the requirement for ERK.