Computational Investigation of the Covalent Inhibition Mechanism of Bruton's Tyrosine Kinase by Ibrutinib.

Covalent inhibitors represent a promising class of therapeutic compounds. Nonetheless, rationally designing covalent inhibitors to achieve a right balance between selectivity and reactivity remains extremely challenging. To better understand the covalent binding mechanism, a computational study is carried out using the irreversible covalent inhibitor of Bruton tyrosine kinase (BTK) ibrutinib as an example. A multi-µs classical molecular dynamics trajectory of the unlinked inhibitor is generated to explore the fluctuations of the compound associated with the kinase binding pocket. Then, the reaction pathway leading to the formation of the covalent bond with the cysteine residue at position 481 via a Michael addition is determined using the string method in collective variables on the basis of hybrid quantum mechanical-molecular mechanical (QM/MM) simulations. The reaction pathway shows a strong correlation between the covalent bond formation and the protonation/deprotonation events taking place sequentially in the covalent inhibition reaction, consistent with a 3-step reaction with transient thiolate and enolates intermediate states. Two possible atomistic mechanisms affecting deprotonation/protonation events from the thiolate to the enolate intermediate were observed: a highly correlated direct pathway involving proton transfer to the Cα of the acrylamide warhead from the cysteine involving one or a few water molecules and a more indirect pathway involving a long-lived enolate intermediate state following the escape of the proton to the bulk solution. The results are compared with experiments by simulating the long-time kinetics of the reaction using kinetic modeling.

Membrane Anchoring of Hck Kinase via the Intrinsically Disordered SH4-U and Length Scale Associated with Subcellular Localization.

Src family kinases (SFKs) are a group of nonreceptor tyrosine kinases that are characterized by their involvement in critical signal transduction pathways. SFKs are often found attached to membranes, but little is known about the conformation of the protein in this environment. Here, solution nuclear magnetic resonance (NMR), neutron reflectometry (NR), and molecular dynamics (MD) simulations were employed to study the membrane interactions of the intrinsically disordered SH4 and Unique domains of the Src family kinase Hck. Through development of a procedure to combine the information from the different techniques, we were able produce a first-of-its-kind atomically detailed structural ensemble of a membrane-bound intrinsically disordered protein. Evaluation of the model demonstrated its consistency with previous work and provided insight into how SFK Unique domains act to differentiate the family members from one another. Fortuitously, the position of the ensemble on the membrane allowed the model to be combined with configurations of the multidomain Hck kinase previously determined from small-angle solution X-ray scattering to produce full-length models of membrane-anchored Hck. The resulting models allowed us to estimate that the kinase active site is positioned about 65 ± 35 Å away from the membrane surface, offering the first estimations of the length scale associated with the concept of SFK subcellular localization.

1H, 15N, and 13C resonance assignments of the intrinsically disordered SH4 and Unique domains of Hck.

Hematopoietic cell kinase (Hck) is an important signaling enzyme and a potential drug target for HIV infections and Bcr/Abl-chronic myeloid leukemia. The protein shares the same SH4-Unique-SH3-SH2-kinase multi-domain architecture as the other eight members of the Src family of non-receptor tyrosine kinases. These enzymes are often found anchored to the intracellular side of the membrane via lipidation of the SH4 domain and are integral components of signaling cascades localized at the cell surface. Despite the detailed structural information available for the SH3, SH2, and kinase domains of Hck, the intrinsically disordered nature of the SH4 and Unique domains has resulted in a lack of information for this important region of the protein that is responsible for membrane association. Here, we report the 1H, 15N and 13C chemical shifts of the Hck SH4-Unique domains at pH 4.5.

Tyrosine Kinase Activation and Conformational Flexibility: Lessons from Src-Family Tyrosine Kinases.

Protein kinases are enzymes that catalyze the covalent transfer of the γ-phosphate of an adenosine triphosphate (ATP) molecule onto a tyrosine, serine, threonine, or histidine residue in the substrate and thus send a chemical signal to networks of downstream proteins. They are important cellular signaling enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Unregulated protein kinase activity is often associated with a wide range of diseases, therefore making protein kinases major therapeutic targets. A prototypical system of central interest to understand the regulation of kinase activity is provided by tyrosine kinase c-Src, which belongs to the family of Src-related non-receptor tyrosine kinases (SFKs). Although the broad picture of autoinhibition via the regulatory domains and via the phosphorylation of the C-terminal tail is well characterized from a structural point of view, a detailed mechanistic understanding at the atomic-level is lacking. Advanced computational methods based on all-atom molecular dynamics (MD) simulations are employed to advance our understanding of tyrosine kinase activation. The computational studies suggest that the isolated kinase domain (KD) is energetically most favorable in the inactive conformation when the activation loop (A-loop) of the KD is not phosphorylated. The KD makes transient visits to a catalytically competent active-like conformation. The process of bimolecular trans-autophosphorylation of the A-loop eventually locks the KD in the active state. Activating point mutations may act by slightly increasing the population of the active-like conformation, enhancing the availability of the A-loop to be phosphorylated. The Src-homology 2 (SH2) and Src-homology 3 (SH3) regulatory domains, depending upon their configuration, either promote the inactive or the active state of the kinase domain. In addition to the roles played by the SH3, SH2, and KD, the Src-homology 4-Unique domain (SH4-U) region also serves as a key moderator of substrate specificity and kinase function. Thus, a fundamental understanding of the conformational propensity of the SH4-U region and how this affects the association to the membrane surface are likely to lead to the discovery of new intermediate states and alternate strategies for inhibition of kinase activity for drug discovery. The existence of a multitude of KD conformations poses a great challenge aimed at the design of specific inhibitors. One promising computational strategy to explore the conformational flexibility of the KD is to construct Markov state models from aggregated MD data.

Facile heme vinyl posttranslational modification in a hemoglobin.

Iron-protoporphyrin IX, or b heme, is utilized as such by a large number of proteins and enzymes. In some cases, notably the c-type cytochromes, this group undergoes a posttranslational covalent attachment to the polypeptide chain, which adjusts the physicochemical properties of the holoprotein. The hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 (GlbN), contrary to the archetypical hemoglobin, modifies its b heme covalently. The posttranslational modification links His117, a residue that does not coordinate the iron, to the porphyrin 2-vinyl substituent and forms a hybrid b/c heme. The reaction is an electrophilic addition that occurs spontaneously in the ferrous state of the protein. This apparently facile type of heme modification has been observed in only two cyanobacterial GlbNs. To explore the determinants of the reaction, we examined the behavior of Synechocystis GlbN variants containing a histidine at position 79, which is buried against the porphyrin 4-vinyl substituent. We found that L79H/H117A GlbN bound the heme weakly but nevertheless formed a cross-link between His79 Nε2 and the heme 4-Cα. In addition to this linkage, the single variant L79H GlbN also formed the native His117-2-Cα bond yielding an unprecedented bis-alkylated protein adduct. The ability to engineer the doubly modified protein indicates that the histidine-heme modification in GlbN is robust and could be engineered in different local environments. The rarity of the histidine linkage in natural proteins, despite the ease of reaction, is proposed to stem from multiple sources of negative selection.

3-Fluorotyrosine as a complementary probe of hemoglobin structure and dynamics: a (19)F-NMR study of Synechococcus sp. PCC 7002 GlbN.

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 (GlbN) contains three tyrosines (Tyr5, Tyr22, and Tyr53), each of which undergoes a structural rearrangement when the protein binds an exogenous ligand such as cyanide. We explored the use of 3-fluorotyrosine and (19)F-NMR spectroscopy for the characterization of GlbN. Assignment of (19)F resonances in fluorinated GlbN (GlbN*) was achieved with individual Tyr5Phe and Tyr53Phe replacements. We observed marked variations in chemical shift and linewidth reflecting the dependence of structural and dynamic properties on oxidation state, ligation state, and covalent attachment of the heme group. The isoelectronic complexes of ferric GlbN* with cyanide and ferrous GlbN* with carbon monoxide gave contrasting spectra, the latter exhibiting heterogeneity and enhanced internal motions on a microsecond-to-millisecond time scale. The strength of the H-bond network involving Tyr22 (B10) and bound cyanide was tested at high pH. 3-Fluorotyrosine at position 22 had a pK(a) value at least 3 units higher than its intrinsic value, 8.5. In addition, evidence was found for long-range communication among the tyrosine sites. These observations demonstrated the utility of the 3-fluorotyrosine approach to gain insight in hemoglobin properties.

Influence of heme post-translational modification and distal ligation on the backbone dynamics of a monomeric hemoglobin.

The cyanobacterium Synechococcus sp. PCC 7002 uses a hemoglobin of the truncated lineage (GlbN) in the detoxification of reactive species generated in the assimilation of nitrate. In view of a sensing or enzymatic role, several states of GlbN are of interest with respect to its structure-activity relationship. Nuclear magnetic resonance spectroscopy was applied to compare the structure and backbone dynamics of six GlbN forms differing in their oxidation state [Fe(II) or Fe(III)], distal ligand to the iron (histidine, carbon monoxide, or cyanide), or heme post-translational modification (b heme or covalently attached heme). Structural properties were assessed with pseudocontact shift calculations. (15)N relaxation data were analyzed by reduced spectral density mapping (picosecond to nanosecond motions) and by inspection of elevated R(2) values (microsecond to millisecond motions). On the picosecond to nanosecond time scale, GlbN exhibited little flexibility and was unresponsive to the differences among the various forms. Regions of slightly higher mobility were the CE turn, the EF loop, and the H-H' kink. In contrast, fluctuations on the microsecond to millisecond time scale depended on the form. Cyanide binding to the ferric state did not enhance motions, whereas reduction to the ferrous bis-histidine state resulted in elevated R(2) values for several amides. This response was attributed, at least in part, to a weakening of the distal histidine coordination. Carbon monoxide binding quenched some of these fluctuations. The results emphasized the role of the distal ligand in dictating backbone flexibility and illustrated the multiple ways in which motions are controlled by the hemoglobin fold.

Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002.

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539-552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10(2)-10(3) M(-1) s(-1) and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.

Structural properties of 2/2 hemoglobins: the group III protein from Helicobacter hepaticus.

The ε-proteobacterium Helicobacter hepaticus (Hh) contains a gene coding for a hemoglobin (Hb). The protein belongs to the 2/2 Hb lineage and is representative of group III, a set of Hbs about which little is known. An expression and purification procedure was developed for Hh Hb. Electronic absorption and nuclear magnetic resonance (NMR) spectra were used to characterize ligation states of the ferric and ferrous protein. The pK(a) of the acid/alkaline transition of ferric Hh Hb was 7.3, an unusually low value. NMR analysis of the cyanomet complex showed the orientation of the heme group to be reversed when compared with most group I and group II 2/2 Hbs. Ferrous Hh Hb formed a stable cyanide complex that yielded NMR spectra similar to those of the carbonmonoxy complex. All forms of Hh Hb were self-associated at NMR concentrations. Comparison was made to the related Campylobacter jejuni 2/2 Hb (Ctb), and the amino acid conservation pattern of group III was reinspected to help in the generalization of structure-function relationships.

Chemical reactivity of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 hemoglobins: covalent heme attachment and bishistidine coordination.

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous H(2)O(2).

Functional and structural characterization of the 2/2 hemoglobin from Synechococcus sp. PCC 7002.

Cyanobacterium Synechococcus sp. PCC 7002 contains a single gene (glbN) coding for GlbN, a protein of the 2/2 hemoglobin lineage. The precise function of GlbN is not known, but comparison to similar 2/2 hemoglobins suggests that reversible dioxygen binding is not its main activity. In this report, the results of in vitro and in vivo experiments probing the role of GlbN are presented. Transcription profiling indicated that glbN is not strongly regulated under any of a large number of growth conditions and that the gene is probably constitutively expressed. High levels of nitrate, used as the sole source of nitrogen, and exposure to nitric oxide were tolerated better by the wild-type strain than a glbN null mutant, whereas overproduction of GlbN in the null mutant background restored the wild-type growth. The cellular contents of reactive oxygen/nitrogen species were elevated in the null mutant under all conditions and were highest under NO challenge or in the presence of high nitrate concentrations. GlbN overproduction attenuated these contents significantly under the latter conditions. The analysis of cell extracts revealed that the heme of GlbN was covalently bound to overproduced GlbN apoprotein in cells grown under microoxic conditions. A peroxidase assay showed that purified GlbN does not possess significant hydrogen peroxidase activity. It was concluded that GlbN protects cells from reactive nitrogen species that could be encountered naturally during growth on nitrate or under denitrifying conditions. The solution structure of covalently modified GlbN was determined and used to rationalize some of its chemical properties.

(1)H, (15)N, and (13)C resonance assignments of the 2/2 hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 in the ferric bis-histidine state.

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 is a monomeric 123-residue Group I 2/2 hemoglobin. Here, we report (1)H, (15)N, and (13)C assignments for the ferric (low-spin, S = (1/2)) protein with a b heme cofactor and after post-translational modification leading to a c-like heme.

Structural and thermodynamic encoding in the sequence of rat microsomal cytochrome b(5).

The water-soluble domain of rat microsomal cytochrome b(5) is a convenient protein with which to inspect the connection between amino acid sequence and thermodynamic properties. In the absence of its single heme cofactor, cytochrome b(5) contains a partially folded stretch of 30 residues. This region is recognized as prone to disorder by programs that analyze primary structures for such intrinsic features. The cytochrome was subjected to amino acid replacements in the folded core (I12A), in the portion that refolds only when in contact with the heme group (N57P), and in both (F35H/H39A/L46Y). Despite the difficulties associated with measuring thermodynamic quantities for the heme-bound species, it was possible to rationalize the energetic consequences of both types of replacements and test a simple equation relating apoprotein and holoprotein stability. In addition, a phenomenological relationship between the change in T(m) (the temperature at the midpoint of the thermal transition) and the change in thermodynamic stability determined by chemical denaturation was observed that could be used to extend the interpretation of incomplete holoprotein stability data. Structural information was obtained by nuclear magnetic resonance spectroscopy toward an atomic-level analysis of the effects.

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins.

The binding of a cofactor to a protein matrix often involves a reorganization of the polypeptide structure. b Hemoproteins provide multiple examples of this behavior. In this minireview, selected monomeric and single b heme proteins endowed with distinct topological properties are inspected for the extent of induced refolding upon heme binding. To complement the data reported in the literature, original results are presented on a two-on-two globin of cyanobacterial origin (Synechococcus sp. PCC 7002 GlbN) and on the heme-containing module of FixL, an oxygen-sensing protein with the mixed alpha/beta topology of PAS domains. GlbN had a stable apoprotein that was further stabilized and locally refolded by heme binding; in contrast, apoFixLH presented features of a molten globule. Sequence analyses (helicity, disorder, and polarity) and solvent accessibility calculations were performed to identify trends in the architecture of b hemoproteins. In several cases, the primary structure appeared biased toward a partially disordered binding pocket in the absence of the cofactor.