Synthesis of the phenylpyridal scaffold as a helical peptide mimetic.

Phenylpyridal- and phenyldipyridal-based scaffolds have been designed and synthesized as novel helical peptide mimetics. The synthesis required optimisation and selective alkylation in producing 2,6-functionalized 3-hydroxypyridine derivatives for a convergent scheme. The pyridine analogues were coupled by a series of Suzuki/Stille types cross-coupling reactions. A series of biaryl and ter-aryl substituted heterocycles were produced. The synthetic approach was concise and high yielding allowing large variability at the wanted side-chain attachment points. A number of compounds were synthesised to show the versatility of the strategy.

High-resolution crystal structures of protein helices reconciled with three-centered hydrogen bonds and multipole electrostatics.

Theoretical and experimental evidence for non-linear hydrogen bonds in protein helices is ubiquitous. In particular, amide three-centered hydrogen bonds are common features of helices in high-resolution crystal structures of proteins. These high-resolution structures (1.0 to 1.5 Å nominal crystallographic resolution) position backbone atoms without significant bias from modeling constraints and identify Φ = -62°, ψ = -43 as the consensus backbone torsional angles of protein helices. These torsional angles preserve the atomic positions of α-ß carbons of the classic Pauling α-helix while allowing the amide carbonyls to form bifurcated hydrogen bonds as first suggested by Némethy et al. in 1967. Molecular dynamics simulations of a capped 12-residue oligoalanine in water with AMOEBA (Atomic Multipole Optimized Energetics for Biomolecular Applications), a second-generation force field that includes multipole electrostatics and polarizability, reproduces the experimentally observed high-resolution helical conformation and correctly reorients the amide-bond carbonyls into bifurcated hydrogen bonds. This simple modification of backbone torsional angles reconciles experimental and theoretical views to provide a unified view of amide three-centered hydrogen bonds as crucial components of protein helices. The reason why they have been overlooked by structural biologists depends on the small crankshaft-like changes in orientation of the amide bond that allows maintenance of the overall helical parameters (helix pitch (p) and residues per turn (n)). The Pauling 3.6(13) α-helix fits the high-resolution experimental data with the minor exception of the amide-carbonyl electron density, but the previously associated backbone torsional angles (Φ, Ψ) needed slight modification to be reconciled with three-atom centered H-bonds and multipole electrostatics. Thus, a new standard helix, the 3.6(13/10)-, Némethy- or N-helix, is proposed. Due to the use of constraints from monopole force fields and assumed secondary structures used in low-resolution refinement of electron density of proteins, such structures in the PDB often show linear hydrogen bonding.

Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo.

Foxp3(+) CD4(+) T helper cells called regulatory T (T reg) cells play a key role in controlling reactivity to self-antigens and onset of autoimmunity. T reg cells either arise in thymus and are called natural T reg (nT reg) cells or are generated in the periphery through induction of Foxp3 and are called inducible T reg (iT reg) cells. The relative contributions of iT reg cells and nT reg cells in peripheral tolerance remain unclear as a result of an inability to separate these two subsets of T reg cells. Using a combination of novel TCR transgenic mice with a defined self-antigen specificity and conventional mouse models, we demonstrate that a cell surface molecule, neuropilin-1 (Nrp-1), is expressed at high levels on nT reg cells and can be used to separate nT reg versus iT reg cells in certain physiological settings. In addition, iT reg cells generated through antigen delivery or converted under homeostatic conditions lack Nrp-1 expression. Nrp-1(lo) iT reg cells show similar suppressive activity to nT reg cells in controlling ongoing autoimmune responses under homeostatic conditions. In contrast, their activity might be compromised in certain lymphopenic settings. Collectively, our data show that Nrp-1 provides an excellent marker to distinguish distinct T reg subsets and will be useful in studying the role of nT reg versus iT reg cells in different disease settings.

Chemogenomics with protein secondary-structure mimetics.

During molecular recognition of proteins in biological systems, helices, reverse turns, and beta-sheets are dominant motifs. Often there are therapeutic reasons for blocking such recognition sites, and significant progress has been made by medicinal chemists in the design and synthesis of semirigid molecular scaffolds on which to display amino acid side chains. The basic premise is that preorganization of the competing ligand enhances the binding affinity and potential selectivity of the inhibitor. In this chapter, current progress in these efforts is reviewed.

Back to the future: ribonuclease A.

Pancreatic ribonuclease A (EC 3.1.27.5, RNase) is, perhaps, the best-studied enzyme of the 20th century. It was isolated by René Dubos, crystallized by Moses Kunitz, sequenced by Stanford Moore and William Stein, and synthesized in the laboratory of Bruce Merrifield, all at the Rockefeller Institute/University. It has proven to be an excellent model system for many different types of experiments, both as an enzyme and as a well-characterized protein for biophysical studies. Of major significance was the demonstration by Chris Anfinsen at NIH that the primary sequence of RNase encoded the three-dimensional structure of the enzyme. Many other prominent protein chemists/enzymologists have utilized RNase as a dominant theme in their research. In this review, the history of RNase and its offspring, RNase S (S-protein/S-peptide), will be considered, especially the work in the Merrifield group, as a preface to preliminary data and proposed experiments addressing topics of current interest. These include entropy-enthalpy compensation, entropy of ligand binding, the impact of protein modification on thermal stability, and the role of protein dynamics in enzyme action. In continuing to use RNase as a prototypical enzyme, we stand on the shoulders of the giants of protein chemistry to survey the future.

Validated ligand mapping of ACE active site.

Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 A, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.