Krishna Saxena

NMR analysis of a tau phosphorylation pattern

The phosphorylation of the neuronal Tau protein modulates both its physiological role of microtubule binding and its aggregation into paired helical fragments observed in Alzheimers diseased neurons. However, detailed knowledge of the role of phosphorylation at specific sites has been hampered by the analytical difficulties to evaluate the level of site-specific phosphate incorporation. Even with recombinant kinases, mass spectrometry and immunodetection are not evident for determining the full phosphorylation pattern in a qualitative and quantitative manner. We show here that heteronuclear NMR spectroscopy on a 15N labeled Tau sample modified by the cAMP dependent kinase allows identification of all phosphorylation sites, measures their level of phosphate integration, and yields kinetic data for the enzymatic modification of the individual sites. Filtering through the 15N label discards the necessity of any further sample purification and allows the in situ monitoring of kinase activity at selected sites. We finally demonstrate that the NMR approach can equally be used to evaluate potential kinase inhibitors in a straightforward manner.

Polo-like Kinase 1-mediated Phosphorylation Stabilizes Pin1 by Inhibiting Its Ubiquitination in Human Cells

The Polo-like kinase 1 (Plk1) is a key regulator of mitosis. It is reported that the human peptidyl-prolyl cis/trans-isomerase Pin1 binds to Plk1 from mitotic cell extracts in vitro. Here we demonstrate that Ser-65 in Pin1 is the major site for Plk1-specific phosphorylation, and the polo-box domain of Plk1 is required for this phosphorylation. Interestingly, the phosphorylation of Pin1 by Plk1 does not affect its isomerase activity but rather is linked to its protein stability. Pin1 is ubiquitinated in HeLa S3 cells, and substitution of Glu for Ser-65 reduces the ubiquitination of Pin1. Furthermore, inhibition of Plk1 activity by expression of a dominant negative form of Plk1 or by transfection of small interfering RNA targeted to Plk1 enhances the ubiquitination of Pin1 and subsequently reduces the amount of Pin1 in human cancer cells. Since previous reports suggested that Plk1 is a substrate of Pin1, our work adds a new dimension to this interaction of two important mitotic regulators.

Molecular Mechanism of SSR128129E, an Extracellularly Acting, Small-Molecule, Allosteric Inhibitor of FGF Receptor Signaling

The fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling network plays an important role in cell growth, survival, differentiation, and angiogenesis. Deregulation of FGFR signaling can lead to cancer development. Here, we report an FGFR inhibitor, SSR128129E (SSR), that binds to the extracellular part of the receptor. SSR does not compete with FGF for binding to FGFR but inhibits FGF-induced signaling linked to FGFR internalization in an allosteric manner, as shown by crystallography studies, nuclear magnetic resonance, Fourier transform infrared spectroscopy, molecular dynamics simulations, free energy calculations, structure-activity relationship analysis, and FGFR mutagenesis. Overall, SSR is a small molecule allosteric inhibitor of FGF/FGFR signaling, acting via binding to the extracellular part of the FGFR.

Spectroscopic, molecular modeling, and NMR-spectroscopic investigation of the binding mode of the natural alkaloids berberine and sanguinarine to human telomeric G-quadruplex DNA.

G-quadruplex structures can be formed at the single-stranded overhang of telomeric DNA, and ligands able to stabilize this structure have recently been identified as potential anticancer drugs. Among the potential G-quadruplex binders, we have studied the binding ability of berberine and sanguinarine, two members of the alkaloid family, an important class of natural products long known for medicinal purpose. Our spectroscopic (CD, NMR, and fluorescence) studies and molecular modeling approaches revealed binding modes at ligand-complex stoichiometries >1:1 and ligand self-association induced by DNA for the interactions of the natural alkaloids berberine and sanguinarine with the human telomeric G-quadruplex DNA.

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Significance Channelrhodopsin-2 is a dimeric membrane protein functioning as a light-gated ion channel, which has triggered numerous optogenetic applications. We present the first NMR study, to our knowledge, by which structural details of the retinal cofactor could be resolved. This study was only possible by enhancing the detection sensitivity 60-fold through dynamic nuclear polarization (DNP), a highly promising hybrid method linking EPR with solid-state NMR spectroscopy. Our data show that ground-state channelrhodopsin-2 contains the retinal cofactor in its all-trans configuration with a slightly perturbed polyene chain. Three different photointermediates could be trapped and analyzed. Our study shows that DNP-enhanced solid-state NMR is a key method for bridging the gap between X-ray–based structure analysis and functional studies toward a highly resolved molecular picture. Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15N-labeled channelrhodopsin-2 carrying 14,15-13C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P1500 K-like state, the slowly decaying late intermediate P4480, and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray–based structure analysis, which is required for resolving molecular mechanisms.

NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: application to the catalytic subunit of cAMP-dependent protein kinase.

Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best‐characterized protein kinases is the catalytic subunit of cAMP‐dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply (2H,13C,15N) labeled protein and classical NMR assignment experiments, 2) back‐calculation of chemical shifts from known X‐ray structures, 3) use of paramagnetic adenosine derivatives as spin‐labels, and 4) selective amino acid labeling. Interpretation of chemical‐shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D 1H,15N TROSY of an inactive Thr197Ala mutant with the wild‐type enzyme.

The structure of porin from Paracoccus denitrificans at 3.1 A resolution

© 1997 Federation of European Biochemical Societies.

Discovery of Mycobacterium Tuberculosis Protein Tyrosine Phosphatase A (MptpA) Inhibitors Based on Natural Products and a Fragment-Based Approach

Protein phosphorylation and dephosphorylation reactions are at the heart of innumerable biological processes. Aberrant protein phosphorylation contributes to the development of many human diseases including cancer and diabetes. Due to this biological importance, protein kinases, which catalyse protein phosphorylation, and their antagonists, protein phosphatases (PPs), have moved into the focus of a rapidly growing number of medicinal-chemistry and chemicalbiology research programs. Several bacterial pathogens produce eukaryotic-like protein phosphatases that have been implicated in virulence. A particularly important case is Myobacterium tuberculosis, which is the causative agent of tuberculosis (TB) and a major cause of mortality around the world. M. tuberculosis has two functional phosphatases, MptpA and MptpB. These enzymes are secreted by growing mycobacterial cells. They are believed to mediate mycobacterial survival in host cells by dephosphorylating proteins that are involved in interferon-g signaling pathways. About one third of the world’s population is infected with M. tuberculosis, and there is an increasing spread of drug-resistant mycobacteria. Therefore, there is a growing need for the development of new therapeutic agents for the treatment of tuberculosis. In the light of this urgent demand, the Mptps have been proposed as new potential anti-TB drug targets. However, to date, inhibitors of these enzymes have not been described. Here we describe the discovery of MptpA inhibitors by two different and complementary approaches for the identification of initial hits in screening collections, namely natural-productinspired and fragment-based library development. We have previously forwarded the notion that biologically active natural products should be regarded as evolutionarily selected and biologically prevalidated starting points for inhibitor development. Based on this principle and the fact that MptpA is a tyrosine phosphatase, we have investigated whether natural products and their analogues that have already served as guiding structures for the discovery of new classes of phosphatase inhibitors could be employed for the identification of the first Mptp inhibitors. Initially the stevastelins (Scheme 1) were considered as possible starting points for the development of MptpA inhibitors.

How Much NMR Data Is Required To Determine a Protein–Ligand Complex Structure?

Here we present an NMR‐based approach to solving protein–ligand structures. The procedure is guided by biophysical, biochemical, or knowledge‐based data. The structures are mainly derived from ligand‐induced chemical‐shift perturbations (CSP) induced in the resonances of the protein and ligand‐detected saturated transfer difference signals between ligands and selectively labeled proteins (SOS‐NMR). Accuracy, as judged by comparison with X‐ray results, depends on the nature and completeness of the experimental data. An experimental protocol is proposed that starts with calculations that make use of readily available chemical‐shift perturbations as experimental constraints. If necessary, more sophisticated experimental results have to be added to improve the accuracy of the protein–ligand complex structure. The criteria for evaluation and selection of meaningful complex structures are discussed. These are exemplified for three complexes, and we show that the approach bridges the gap between theoretical docking approaches and complex NMR schemes for determining protein–ligand complexes; especially for relatively weak binders that do not lead to intermolecular NOEs.

Biomolecular NMR: a chaperone to drug discovery

Biomolecular NMR now contributes routinely to every step in the development of new chemical entities ahead of clinical trials. The versatility of NMR — from detection of ligand binding over a wide range of affinities and a wide range of drug targets with its wealth of molecular information, to metabolomic profiling, both ex vivo and in vivo — has paved the way for broadly distributed applications in academia and the pharmaceutical industry. Proteomics and initial target selection both benefit from NMR: screenings by NMR identify lead compounds capable of inhibiting protein–protein interactions, still one of the most difficult development tasks in drug discovery. NMR hardware improvements have given access to the microgram domain of phytochemistry, which should lead to the discovery of novel bioactive natural compounds. Steering medicinal chemists through the lead optimisation process by providing detailed information about protein–ligand interactions has led to impressive success in the development of novel drugs. The study of biofluid composition — metabonomics — provides information about pharmacokinetics and helps toxicological safety assessment in animal model systems. In vivo, magnetic resonance spectroscopy interrogates metabolite distributions in living cells and tissues with increasing precision, which significantly impacts the development of anticancer or neurological disorder therapeutics. An overview of different steps in recent drug discovery is presented to illuminate the links with the most recent advances in NMR methodology.