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Src family kinases (SFKs) are a group of non-receptor 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 multi-domain 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 lengthscale associated with the concept of SFK subcellular localization.

Hemopoietic 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 8 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 (1)H, (15)N and (13)C chemical shifts of the Hck SH4-Unique domains at pH 4.5.

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.

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.

The Src family kinases (SFKs) are a group of nine non-receptor tyrosine kinases known for their involvement in critical signal transduction pathways. Structurally, SFKs share a common multi-domain architecture, consisting of SH3, SH2, and catalytic (SH1) domains attached to a membrane-anchoring SH4 domain through an intrinsically disordered ∼80 residue region of low sequence conservation called the Unique (U) domain. Despite the wealth of information on SFK structure and function, little is known about the nature of these enzymes in the membrane-associated form. Interestingly, SH4-U domains in different species share common sequence features, despite differences between SFK members, arguing for their importance in differentiating the functions of SFKs and raising questions about their ability to participate in SFK signaling.Hematopoetic cell kinase (Hck), a phagocyte specific proto-oncogene member of the SFKs, is a potential drug target for HIV infections and Bcr/Abl-chronic myeloid leukemia. To achieve structural characterization of the membrane-associated Hck via its SH4-U domains, data from solution and solid-state NMR, neutron reflection, and fluorescence resonance energy transfer were computationally integrated using a novel maximum-entropy multi-resolution “restrained-ensemble molecular dynamics” (reMD) simulation scheme. This framework has allowed us to detail features of the membrane-protein complex at atomic resolution and identify possible new constraints on Hck's ability to phosphorylate downstream targets. In vivo assays are being developed to test the relevance of these findings.

The e-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 pKa 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. © 2011 IUBMB IUBMB Life, 63(3): 197–205, 2011

The water-soluble domain of rat microsomal cytochrome b5 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 b5 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 Tm (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. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 428–442, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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.

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).

Src family kinases (SFKs) are a group of nine non-receptor tyrosine kinases that play critical roles in cellular transduction pathways. These highly sought after therapeutic targets are prevalent and promiscuous; therefore, they must be tightly regulated to prevent unchecked signaling cascades. Inter-domain interactions that hinder kinase function are key SFK regulatory mechanisms, and detailed information about these interactions is integral to understanding how these enzymes function.Hck is a SFK primarily found in cells of hematopoietic lineage and is among the most well studied members of the family. Crystal structures of Hck in the down-regulated form and solution studies detailing catalytic responses to regulatory domain displacement provide a robust framework for characterizing the regulatory interactions. Adding to this knowledge, we provide fundamental information on Hck regulation by measuring binding free energies of the native domain-peptide interactions. A combined experimental and computational approach was used to complement and expand the usefulness of the data. The results were compared with bulk FRET measurements made using the full-length kinase to observe how these interactions are modified in the context of the protein.Given the high degree of homology among family members, it is unsurprising that SFKs have some redundant or compensatory functions. However, it has been shown that even the most closely related members of the family cannot always substitute functionally for one another in vivo and the differences responsible have yet to be fully elucidated. Our results lay the groundwork for comparative analysis between different family members and are expected to aid in identifying features that distinguish these enzymes from one another.

The hemoglobin of the cyanobacterium Synechococcus sp. PCC 7002 (GlbN) protects the cell from reactive oxygen/nitrogen species.1 GlbN coordinates the heme group with two histidines in the absence of an exogenous ligand and undergoes an unusual post-translational attachment of the heme group to the H helix. GlbN is well behaved by NMR spectroscopic standards, making it an excellent target for structural and dynamic studies aimed at characterizing the differential lability of the axial histidines, the perturbations caused by the post-translational modification, and the effect of exogenous ligand binding. The structure of GlbN is largely unperturbed by the covalent heme-protein cross-link,2 but binding of diatomic ligands such as CO (to ferrous GlbN) and CN- (to ferric GlbN) induces the formation of a distal hydrogen bond network and causes a shift of the B and E helices. 15N relaxation measurements indicate that the differences in ps-ns dynamics between the proximal and distal sides of the heme cofactor are minimal and also independent of heme covalent attachment. Upon CO or CN- binding the us-ms timescale motions are enhanced in the B and E helices, suggesting that the preferential displacement of the distal histidine is due to the stability of the final bound state rather than an intrinsic bond strength difference. The implications of increased dynamics on the distal side of the pocket after ligand binding will be discussed in terms of ligand migration inside GlbN and related globins.1) Scott et al., Biochemistry, 2010, 49: 7000-7011.2) Pond et al., Biomolecular NMR Assignments, 2009, 3: 211-214.Supported by NSF grant MCB 0843439.