Monitoring Ras Interactions with the Nucleotide Exchange Factor Sos using Site-specific NMR Reporter Signals and Intrinsic Fluorescence

The activity of Ras is controlled by the inter-conversion between GTP- and GDP-bound forms, partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly [15N]-labeled Ras, as well as [13C-methyl-M,I]-labeled Sos, for observing site-specific details of Ras:Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP, or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized, by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants, as well as their selected functional mutants, was also investigated using intrinsic fluorescence. The data supports a positive feedback activation of Sos by Ras•GTP, with Ras•GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras•GDP, suggesting that Sos should actively promote unidirectional GDP→GTP exchange on Ras, in preference of passive homonucleotide exchange. Ras•GDP weakly binds to the catalytic, but not to the allosteric site of Sos. This confirms that Ras•GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras:Sos interactions

MIPModDB: a central resource for the superfamily of major intrinsic proteins

The channel proteins belonging to the major intrinsic proteins (MIP) superfamily are diverse and are found in all forms of life. Water-transporting aquaporin and glycerol-specific aquaglyceroporin are the prototype members of the MIP superfamily. MIPs have also been shown to transport other neutral molecules and gases across the membrane. They have internal homology and possess conserved sequence motifs. By analyzing a large number of publicly available genome sequences, we have identified more than 1000 MIPs from diverse organisms. We have developed a database MIPModDB which will be a unified resource for all MIPs. For each MIP entry, this database contains information about the source, gene structure, sequence features, substitutions in the conserved NPA motifs, structural model, the residues forming the selectivity filter and channel radius profile. For selected set of MIPs, it is possible to derive structure-based sequence alignment and evolutionary relationship. Sequences and structures of selected MIPs can be downloaded from MIPModDB database which is freely available at http://bioinfo.iitk.ac.in/MIPModDB

Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase.

Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp-Phe-Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called 'DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a 'DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-β4 loop and 'molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.This work was funded as part of the AstraZeneca Internal Postdoctoral program. All authors with the exception of G.S.T. are employees (and stockholders) of AstraZeneca UK Ltd or MedImmune LLC, or were at the time that this study was conducted.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncomms887

Predicting Inactive Conformations of Protein Kinases Using Active Structures: Conformational Selection of Type-II Inhibitors

Protein kinases have been found to possess two characteristic conformations in their activation-loops: the active DFG-in conformation and the inactive DFG-out conformation. Recently, it has been very interesting to develop type-II inhibitors which target the DFG-out conformation and are more specific than the type-I inhibitors binding to the active DFG-in conformation. However, solving crystal structures of kinases with the DFG-out conformation remains a challenge, and this seriously hampers the application of the structure-based approaches in development of novel type-II inhibitors. To overcome this limitation, here we present a computational approach for predicting the DFG-out inactive conformation using the DFG-in active structures, and develop related conformational selection protocols for the uses of the predicted DFG-out models in the binding pose prediction and virtual screening of type-II ligands. With the DFG-out models, we predicted the binding poses for known type-II inhibitors, and the results were found in good agreement with the X-ray crystal structures. We also tested the abilities of the DFG-out models to recognize their specific type-II inhibitors by screening a database of small molecules. The AUC (area under curve) results indicated that the predicted DFG-out models were selective toward their specific type-II inhibitors. Therefore, the computational approach and protocols presented in this study are very promising for the structure-based design and screening of novel type-II kinase inhibitors

Allosteric Interactions between the Myristate- and ATP-Site of the Abl Kinase

Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors

A theoretical entropy score as a single value to express inhibitor selectivity

<p>Abstract</p> <p>Background</p> <p>Designing maximally selective ligands that act on individual targets is the dominant paradigm in drug discovery. Poor selectivity can underlie toxicity and side effects in the clinic, and for this reason compound selectivity is increasingly monitored from very early on in the drug discovery process. To make sense of large amounts of profiling data, and to determine when a compound is sufficiently selective, there is a need for a proper quantitative measure of selectivity.</p> <p>Results</p> <p>Here we propose a new theoretical entropy score that can be calculated from a set of IC₅₀data. In contrast to previous measures such as the 'selectivity score', Gini score, or partition index, the entropy score is non-arbitary, fully exploits IC₅₀data, and is not dependent on a reference enzyme. In addition, the entropy score gives the most robust values with data from different sources, because it is less sensitive to errors. We apply the new score to kinase and nuclear receptor profiling data, and to high-throughput screening data. In addition, through analyzing profiles of clinical compounds, we show quantitatively that a more selective kinase inhibitor is not necessarily more drug-like.</p> <p>Conclusions</p> <p>For quantifying selectivity from panel profiling, a theoretical entropy score is the best method. It is valuable for studying the molecular mechanisms of selectivity, and to steer compound progression in drug discovery programs.</p

Mutation D816V Alters the Internal Structure and Dynamics of c-KIT Receptor Cytoplasmic Region: Implications for Dimerization and Activation Mechanisms

The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites

Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor

G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography1–3, the details of ligand induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR4, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained 5–9. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey β1-adrenergic receptor (β1AR) 10–12. Labelling with [15N] valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental method to delineate signal transmission networks at high resolution in GPCRs

Residual dipolar couplings in short peptides reveal systematic conformational preferences of individual amino acids

Residual dipolar couplings (RDCs) observed by NMR in solution under weak alignment conditions can monitor average net orientations and order parameters of individual bonds. By their simple geometrical dependence, RDCs bear particular promise for the quantitative characterization of conformations in partially folded or unfolded proteins. We have systematically investigated the influence of amino acid substitutions X on the conformation of unfolded model peptides EGAAXAASS as monitored by their (1)H(Nu)-(15)N and (1)H(alpha)-(13)C(alpha) RDCs detected at natural abundance of (15)N and (13)C in strained polyacrylamide gels. In total, 14 single amino acid substitutions were investigated. The RDCs show a specific dependence on the substitution X that correlates to steric or hydrophobic interactions with adjacent amino acids. In particular, the RDCs for the glycine and proline substitutions indicate less or more order, respectively, than the other amino acids. The RDCs for aromatic substitutions tryptophane and tyrosine give evidence of a kink in the peptide backbone. This effect is also observable for orientation by Pf1 phages and corroborated by variations in (13)C(alpha) secondary shifts and (3)J(HNH)(alpha) scalar couplings in isotropic samples. RDCs for a substitution with the beta-turn sequence KNGE differ from single amino acid substitutions. Terminal effects and next neighbor effects could be demonstrated by further specific substitutions. The results were compared to statistical models of unfolded peptide conformations derived from PDB coil subsets, which reproduce overall trends for (1)H(Nu)-(15)N RDCs for most substitutions, but deviate more strongly for (1)H(alpha)-(13)C(alpha) RDCs. The outlined approach opens the possibility to obtain a systematic experimental characterization of the influence of individual amino acid/amino acid interactions on orientational preferences in polypeptides